Background DNA-protein cross-links (DPCs) are one of the most deleterious DNA lesions, originatin... more Background DNA-protein cross-links (DPCs) are one of the most deleterious DNA lesions, originating from various sources, including enzymatic activity. For instance, topoisomerases, which play a fundamental role in DNA metabolic processes such as replication and transcription, can be trapped and remain covalently bound to DNA in the presence of poisons or nearby DNA damage. Given the complexity of individual DPCs, numerous repair pathways have been described. The protein tyrosyl-DNA phosphodiesterase 1 (Tdp1) has been demonstrated to be responsible for removing topoisomerase 1 (Top1). Nevertheless, studies in budding yeast have indicated that alternative pathways involving Mus81, a structure-specific DNA endonuclease, could also remove Top1 and other DPCs. Results This study shows that MUS81 can efficiently cleave various DNA substrates modified by fluorescein, streptavidin or proteolytically processed topoisomerase. Furthermore, the inability of MUS81 to cleave substrates bearing na...
Telomeres-repeated, noncoding nucleotide motifs and associated proteins that are found at the end... more Telomeres-repeated, noncoding nucleotide motifs and associated proteins that are found at the ends of eukaryotic chromosomes-mediate genome stability and determine cellular lifespan 1. Telomeric-repeat-containing RNA (TERRA) is a class of long noncoding RNAs (lncRNAs) that are transcribed from chromosome ends 2,3 ; these RNAs in turn regulate telomeric chromatin structure and telomere maintenance through the telomere-extending enzyme telomerase 4-6 and homology-directed DNA repair 7,8. The mechanisms by which TERRA is recruited to chromosome ends remain poorly defined. Here we develop a reporter system with which to dissect the underlying mechanisms, and show that the UUAGGG repeats of TERRA are both necessary and sufficient to target TERRA to chromosome ends. TERRA preferentially associates with short telomeres through the formation of telomeric DNA-RNA hybrid (R-loop) structures that can form in trans. Telomere association and R-loop formation trigger telomere fragility and are promoted by the recombinase RAD51 and its interacting partner BRCA2, but counteracted by the RNA-surveillance factors RNaseH1 and TRF1. RAD51 physically interacts with TERRA and catalyses R-loop formation with TERRA in vitro, suggesting a direct involvement of this DNA recombinase in the recruitment of TERRA by strand invasion. Together, our findings reveal a RAD51-dependent pathway that governs TERRA-mediated R-loop formation after transcription, providing a mechanism for the recruitment of lncRNAs to new loci in trans. TERRA is transcribed from numerous chromosome ends, and comprises both subtelomeric sequences and telomeric repeats. More than 50% of TERRA is associated with chromatin 9. To investigate how TERRA is recruited to or retained at telomeres, we generated a plasmid encoding 24 copies of the stem-loop of phage PP7 (ref. 10) under the control of the tetracycline-inducible (TET) promoter, followed by 90 TTAGGG repeats (Fig. 1a). To generate full-length TERRA transcripts, we also cloned the human chromosome Xq and 15q subtelomeric regions containing the TERRA start sites between the PP7 stem-loops and the TTAGGG repeats. The constructs were then transiently transfected into HeLa clones that were constitutively expressing the PP7 coat protein fused to GFP (PCP-GFP) and a nuclear-localization signal. PCP-GFP exhibited a diffuse signal in the nucleus but formed nuclear foci upon expression of the PP7 stem-loops, which are bound by PCP and can gather up to 48 PCP-GFP molecules per RNA. These foci did not co-localize with telomeres (Fig. 1b). The fusion of the subtelomeric region of 15q or Xq TERRA to the stem-loops did not promote substantial trafficking of the PP7 foci to telomeres. However, when the telomeric TTAGGG repeats were fused downstream of PP7, co-localization with telomeres occurred, as analysed by conventional and confocal imaging (Fig. 1b), indicating that the 5′-UUAGGG-3′ repeats of TERRA drive telomere association. The full-length PP7-tagged 15q and Xq chimaeric TERRA also showed marked co-localization with telomeres (Fig. 1b and Extended Data Fig. 1a). Therefore, chimaeric TERRAs that originated from a plasmid were directed to telomeres in trans. To eliminate possible confounding effects due to the high plasmid copy number or increased levels of transgenic TERRA, we used CRISPR-Cas9 technology to integrate the chimaeric TERRA constructs into the genome at the adeno-associated-virus integration site 1 (AAVS1) on chromosome 19, which represents a safe harbour for transgene expression 11 (Extended Data Fig. 1b). Following isolation of clones, we confirmed monoallelic site-specific integration of the full constructs by polymerase chain reaction (PCR) and sequencing. These TERRA expression levels were lower than the levels of expression from plasmids, giving one to three foci-indicative of displacement from the transcription site. But, similar to the results obtained upon transient transfection, the PP7 loops formed nuclear foci, and only when fused to 5′-UUAGGG-3′ repeats did the chimaeric RNAs co-localize with telomeres (Extended Data Fig. 1c, d). Shorter telomeres recruit more TERRA In Saccharomyces cerevisiae and Schizosaccharomyces pombe, short telomeres recruit more TERRA, possibly to facilitate telomere maintenance through recombination or telomerase recruitment 5,6,8. To explore the putative roles of telomere length in TERRA recruitment
The proper repair of deleterious DNA lesions such as double strand breaks prevents genomic instab... more The proper repair of deleterious DNA lesions such as double strand breaks prevents genomic instability and carcinogenesis. In yeast, the Rad52 protein mediates DSB repair via homologous recombination. In mammalian cells, despite the presence of the RAD52 protein, the tumour suppressor protein BRCA2 acts as the predominant mediator during homologous recombination. For decades, it has been believed that the RAD52 protein played only a back-up role in the repair of DSBs performing an error-prone single strand annealing (SSA). Recent studies have identified several new functions of the RAD52 protein and have drawn attention to its important role in genome maintenance. Here, we show that RAD52 activities are enhanced by interacting with a small and highly acidic protein called DSS1. Binding of DSS1 to RAD52 changes the RAD52 oligomeric conformation, modulates its DNA binding properties, stimulates SSA activity and promotes strand invasion. Our work introduces for the first time RAD52 as ...
CDK12 is a kinase associated with elongating RNA polymerase II (RNAPII) and is frequently mutated... more CDK12 is a kinase associated with elongating RNA polymerase II (RNAPII) and is frequently mutated in cancer. CDK12 depletion reduces the expression of homologous recombination (HR) DNA repair genes, but comprehensive insight into its target genes and cellular processes is lacking. We use a chemical genetic approach to inhibit analog-sensitive CDK12, and find that CDK12 kinase activity is required for transcription of core DNA replication genes and thus for G1/S progression. RNA-seq and ChIP-seq reveal that CDK12 inhibition triggers an RNAPII processivity defect characterized by a loss of mapped reads from 3 0 ends of predominantly long, poly(A)-signal-rich genes. CDK12 inhibition does not globally reduce levels of RNAPII-Ser2 phosphorylation. However, individual CDK12-dependent genes show a shift of P-Ser2 peaks into the gene body approximately to the positions where RNAPII occupancy and transcription were lost. Thus, CDK12 catalytic activity represents a novel link between regulation of transcription and cell cycle progression. We propose that DNA replication and HR DNA repair defects as a consequence of CDK12 inactivation underlie the genome instability phenotype observed in many cancers.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 2016
Understanding the molecular mechanism of aging could have enormous medical implications. Despite ... more Understanding the molecular mechanism of aging could have enormous medical implications. Despite a century of research, however, there is no universally accepted theory regarding the molecular basis of aging. On the other hand, there is plentiful evidence suggesting that DNA constitutes the central molecule in this process. Here, we review the roles of chromatin structure, DNA damage, and shortening of telomeres in aging and propose a hypothesis for how their interplay leads to aging phenotypes.
Homologous recombination (HR) maintains genome stability by repairing DNA double-strand breaks an... more Homologous recombination (HR) maintains genome stability by repairing DNA double-strand breaks and gaps and restarting replication forks. It is an error-free pathway that uses a homologous sequence in the genome to copy the damaged genetic information. In the present chapter, we will discuss in detail the mechanism by which HR operates to maintain genome stability as revealed by studies predominantly performed in Saccharomyces cerevisiae. We will then discuss the similarities and dissimilarities between yeast and humans while emphasizing the importance of HR in suppressing carcinogenesis and as a potential therapeutic target.
A variety of DNA lesions, secondary DNA structures or topological stress within the DNA template ... more A variety of DNA lesions, secondary DNA structures or topological stress within the DNA template may lead to stalling of the replication fork. Recovery of such forks is essential for the maintenance of genomic stability. The structure-specific endonuclease Mus81-Mms4 has been implicated in processing DNA intermediates that arise from collapsed forks and homologous recombination. According to previous genetic studies, the Srs2 helicase may play a role in the repair of double-strand breaks and ssDNA gaps together with Mus81-Mms4. In this study, we show that the Srs2 and Mus81-Mms4 proteins physically interact in vitro and in vivo and we map the interaction domains within the Srs2 and Mus81 proteins. Further, we show that Srs2 plays a dual role in the stimulation of the Mus81-Mms4 nuclease activity on a variety of DNA substrates. First, Srs2 directly stimulates Mus81-Mms4 nuclease activity independent of its helicase activity. Second, Srs2 removes Rad51 from DNA to allow access of Mus81-Mms4 to cleave DNA. Concomitantly, Mus81-Mms4 inhibits the helicase activity of Srs2. Taken together, our data point to a coordinated role of Mus81-Mms4 and Srs2 in processing of recombination as well as replication intermediates.
Rad54 is an ATP-driven translocase involved in the genome maintenance pathway of homologous recom... more Rad54 is an ATP-driven translocase involved in the genome maintenance pathway of homologous recombination (HR). Although its activity has been implicated in several steps of HR, its exact role(s) at each step are still not fully understood. We have identified a new interaction between Rad54 and the replicative DNA clamp, proliferating cell nuclear antigen (PCNA). This interaction was only mildly weakened by the mutation of two key hydrophobic residues in the highlyconserved PCNA interaction motif (PIP-box) of Rad54 (Rad54-AA). Intriguingly, the rad54-AA mutant cells displayed sensitivity to DNA damage and showed HR defects similar to the null mutant, despite retaining its ability to interact with HR proteins and to be recruited to HR foci in vivo. We therefore surmised that the PCNA interaction might be impaired in vivo and was unable to promote repair synthesis during HR. Indeed, the Rad54-AA mutant was defective in primer extension at the MAT locus as well as in vitro, but additional biochemical analysis revealed that this mutant also had diminished ATPase activity and an inability to promote D-loop formation. Further mutational analysis of the putative PIP-box uncovered that other phenotypically relevant mutants in this domain also resulted in a loss of ATPase activity. Therefore, we have found that although Rad54 interacts with PCNA, the PIP-box motif likely plays only a minor role in stabilizing the PCNA interaction, and rather, this conserved domain is probably an extension of the ATPase domain III.
The biological functions of the small ubiquitin-related Clare Hall Laboratories modifier SUMO app... more The biological functions of the small ubiquitin-related Clare Hall Laboratories modifier SUMO appear to be even more diverse, rang-Blanche Lane ing from nuclear transport to signal transduction, tran-South Mimms scription, and genome stability (Johnson, 2004). At-Herts EN6 3LD tachment of SUMO can induce a relocalization of the United Kingdom target protein within the cell, a conformational change, 2 Max Planck Institute for Terrestrial Microbiology or an alteration of protein-protein interactions, but Karl-von-Frisch-Strasse often its mode of action remains poorly understood. D-35043 Marburg Despite a similar conjugation mechanism, ubiquitin and Germany SUMO generally direct their targets to very different 3 Department of Molecular Biophysics and fates (Gill, 2004). This notion becomes most obvious in Biochemistry situations where the two modifiers compete for the School of Medicine same attachment site on a common substrate protein, Yale University as first noted in the case of the NF-κB inhibitor IκBα, 333 Cedar Street where sumoylation was found to counteract the proteo-New Haven, Connecticut 06520 lytic effect of ubiquitination (Desterro et al., 1998). In the context of DNA replication and repair, SUMO and ubiquitin jointly affect a key signal integrator at the Summary replication fork, PCNA. Besides its function as a sliding clamp that ensures the processivity of replicative DNA Posttranslational modification of proliferating cell polymerases, PCNA serves as a binding platform for nuclear antigen (PCNA), an essential processivity various enzymes involved in DNA repair, chromatin asclamp for DNA polymerases, by ubiquitin and SUMO sembly, and cell cycle control (Jónsson and Hübscher, contributes to the coordination of DNA replication, 1997). In response to DNA-damaging agents, PCNA is damage tolerance, and mutagenesis. Whereas ubiqubiquitinated at a highly conserved lysine (K) residue, uitination in response to DNA damage promotes the K164 (Hoege et al., 2002). In the yeast Saccharomyces bypass of replication-blocking lesions, sumoylation cerevisiae, the same lysine is modified by SUMO during during S phase is damage independent. As both mod-S phase, independent of DNA damage. A second, less ifiers target the same site on PCNA, an antagonistic conserved lysine, K127, is used as a secondary attachaction of SUMO on ubiquitin-dependent DNA damage ment site for SUMO. Ubiquitination of PCNA is critical tolerance has been proposed. We now present evifor the cell's survival in the presence of DNA damage, dence that the apparent negative effect of SUMO on as it facilitates the bypass of replication-blocking lesion bypass is not due to competition with ubiquitilesions. By modification of PCNA, the ubiquitin system nation but is rather mediated by the helicase Srs2p, in fact promotes DNA damage tolerance in two distinct which affects genome stability by suppressing unmodes: whereas monoubiquitination triggers transscheduled homologous recombination. We show that lesion synthesis and damage-induced mutagenesis by Srs2p physically interacts with sumoylated PCNA, error-prone polymerases (Kannouche et al., 2004; which contributes to the recruitment of the helicase Stelter and Ulrich, 2003; Watanabe et al., 2004), polyto replication forks. Our findings suggest a mechaubiquitination activates an error-free damage avoidnism by which SUMO and ubiquitin cooperatively ance pathway that is believed to utilize the genetic incontrol the choice of pathway for the processing of formation encoded by the undamaged sister chromatid DNA lesions during replication. for lesion bypass (Hoege et al., 2002). In the absence of DNA damage, the error-prone polymerase ζ also
The budding yeast Srs2 protein possesses 3 to 5 DNA helicase activity and channels untimely recom... more The budding yeast Srs2 protein possesses 3 to 5 DNA helicase activity and channels untimely recombination to post-replication repair by removing Rad51 from ssDNA. However, it also promotes recombination via a synthesis-dependent strand-annealing pathway (SDSA). Furthermore, at the replication fork, Srs2 is required for fork progression and prevents the instability of trinucleotide repeats. To better understand the multiple roles of the Srs2 helicase during these processes, we analysed the ability of Srs2 to bind and unwind various DNA substrates that mimic structures present during DNA replication and recombination. While leading or lagging strands were efficiently unwound, the presence of ssDNA binding protein RPA presented an obstacle for Srs2 translocation. We also tested the preferred directionality of unwinding of various substrates and studied the effect of Rad51 and Mre11 proteins on Srs2 helicase activity. These biochemical results help us understand the possible role of Srs2 in the processing of stalled or blocked replication forks as a part of post-replication repair as well as homologous recombination (HR).
Homologous recombination plays a key role in the maintenance of genome integrity, especially duri... more Homologous recombination plays a key role in the maintenance of genome integrity, especially during DNA replication and the repair of double-stranded DNA breaks (DSBs). Just a single un-repaired break can lead to aneuploidy, genetic aberrations or cell death. DSBs are caused by a vast number of both endogenous and exogenous agents including genotoxic chemicals or ionizing radiation, as well as through replication of a damaged template DNA or the replication fork collapse. It is essential for cell survival to recognise and process DSBs as well as other toxic intermediates and launch most appropriate repair mechanism. Many helicases have been implicated to play role in these processes, however their detail roles, specificities and co-operativity in the complex protein-protein interaction networks remain unclear. In this review we summarize the current knowledge about Saccharomyces cerevisiae helicase Srs2 and its effect on multiple DNA metabolic processes that generally affect genome stability. It would appear that Srs2 functions as an "Odd-Job Man" in these processes to make sure that the jobs proceed when and where they are needed.
DNA damage tolerance (DDT) and homologous recombination (HR) stabilize replication forks (RFs). R... more DNA damage tolerance (DDT) and homologous recombination (HR) stabilize replication forks (RFs). RAD18/UBC13/three prime repair exonuclease 2 (TREX2)-mediated proliferating cell nuclear antigen (PCNA) ubiquitination is central to DDT, an error-prone lesion bypass pathway. RAD51 is the recombinase for HR. The RAD51 K133A mutation increased spontaneous mutations and stress-induced RF stalls and nascent strand degradation. Here, we report in RAD51 K133A cells that this phenotype is reduced by expressing a TREX2 H188A mutation that
Cdk1 kinase phosphorylates budding yeast Srs2, a member of UvrD protein family that displays both... more Cdk1 kinase phosphorylates budding yeast Srs2, a member of UvrD protein family that displays both DNA translocation and DNA unwinding activities in vitro. Srs2 prevents homologous recombination by dismantling Rad51 filaments and it is also required for double strand break repair. Here we examine the biological significance of Cdk1-dependent phosphorylation of Srs2 using mutants that constitutively express the phosphorylated or unphosphorylated isoforms. We found that Cdk1 targets Srs2 to repair double strand break (DSB) after strand invasion. Srs2 phosphorylation is required to complete synthesis-dependent strand annealing pathway, likely controlling the disassembly of the D-loop intermediate. Cdk1 phosphorylation, indeed, controls the turnover of Srs2 protein at the invading strand, while it is not required for that of Rad51. Further analysis on the recombination phenotypes of the srs2 phospho-mutants indicated that Srs2 phosphorylation is not indeed essential for the removal of to...
Homologous recombination (HR) is a process widespread in nature and essential to maintain the int... more Homologous recombination (HR) is a process widespread in nature and essential to maintain the integrity of the genome via repair of DNA double-strand breaks (DSBs). Rad52 is a DNA-binding protein which plays a key role in HR. It mediates the exchange of the recombination factor RPA associated to single-stranded DNA (ssDNA) by Rad51, resulting in the assembly of the presynaptic filament. Recently, it has been reported that upon DNA damage Rad52 is modified by the small ubiquitin-like modifier (SUMO) protein which shelters Rad52 against proteosomal degradation. As the major SUMOylation sites of Rad52, three lysines (K43, K44, K253) have been identified. The effect of Rad52 DNA binding and interaction with its partners was tested to clarify the regulatory mechanism of Rad52 SUMOylation.
Rothmund-Thomsonův syndrom (RTS) je autozomalni recesivni onemocněni. Typickým klinickým přiznake... more Rothmund-Thomsonův syndrom (RTS) je autozomalni recesivni onemocněni. Typickým klinickým přiznakem RTS je cervena vyražka, znama jako poikiloderma. Pacienti navic trpi ocnim zakalem, kosternimi defekty a vyssi nachylnosti k rakovině kůže a kosti. Mutace v genu RECQ4, ktere byly zjistěny u pacientů s RTS, vedou k produkci nefunkcnich verzi proteinu RecQ4. Domeny proteinu RecQ4 jsou velmi podobne jako u ostatnich clenů RecQ helikaz, avsak jeho přesna funkce je dosud neznama. Nicmeně, protein RecQ4 se podili na udržovani stability geneticke informace, hraje významnou roli při replici a opravě DNA.
Extracellular pH has been assumed to play little if any role in how bacteria respond to antibioti... more Extracellular pH has been assumed to play little if any role in how bacteria respond to antibiotics and antibiotic resistance development. Here, we show that the intracellular pH of Escherichia coli equilibrates to the environmental pH following antibiotic treatment. We demonstrate that this allows the environmental pH to influence the transcription of various DNA damage response genes and physiological processes such as filamentation. Using purified RecA and a known pH-sensitive mutant variant RecA K250R we show how pH can affect the biochemical activity of a protein central to control of the bacterial DNA damage response system. Finally, two different mutagenesis assays indicate that environmental pH affects antibiotic resistance development. Specifically, at environmental pH's greater than six we find that mutagenesis plays a significant role in producing antibiotic resistant mutants. At pH's less than or equal to 6 the genome appears more stable but extensive filamentati...
Dna2 is an essential nuclease-helicase that acts in several distinct DNA metabolic pathways inclu... more Dna2 is an essential nuclease-helicase that acts in several distinct DNA metabolic pathways including DNA replication and recombination. To balance these functions and prevent unscheduled DNA degradation, Dna2 activities must be regulated. Here we show that Saccharomyces cerevisiae Dna2 function is controlled by sumoylation. We map the sumoylation sites to the N-terminal regulatory domain of Dna2 and show that in vitro sumoylation of recombinant Dna2 impairs its nuclease but not helicase activity. In cells, the total levels of the non-sumoylatable Dna2 variant are elevated. However, non-sumoylatable Dna2 shows impaired nuclear localization and reduced recruitment to foci upon DNA damage. Nonsumoylatable Dna2 reduces the rate of DNA end resection, as well as impedes cell growth and cell cycle progression through S phase. Taken together, these findings show that in addition to Dna2 phosphorylation described previously, Dna2 sumoylation is required for the homeostasis of the Dna2 protein function to promote genome stability.
Background DNA-protein cross-links (DPCs) are one of the most deleterious DNA lesions, originatin... more Background DNA-protein cross-links (DPCs) are one of the most deleterious DNA lesions, originating from various sources, including enzymatic activity. For instance, topoisomerases, which play a fundamental role in DNA metabolic processes such as replication and transcription, can be trapped and remain covalently bound to DNA in the presence of poisons or nearby DNA damage. Given the complexity of individual DPCs, numerous repair pathways have been described. The protein tyrosyl-DNA phosphodiesterase 1 (Tdp1) has been demonstrated to be responsible for removing topoisomerase 1 (Top1). Nevertheless, studies in budding yeast have indicated that alternative pathways involving Mus81, a structure-specific DNA endonuclease, could also remove Top1 and other DPCs. Results This study shows that MUS81 can efficiently cleave various DNA substrates modified by fluorescein, streptavidin or proteolytically processed topoisomerase. Furthermore, the inability of MUS81 to cleave substrates bearing na...
Telomeres-repeated, noncoding nucleotide motifs and associated proteins that are found at the end... more Telomeres-repeated, noncoding nucleotide motifs and associated proteins that are found at the ends of eukaryotic chromosomes-mediate genome stability and determine cellular lifespan 1. Telomeric-repeat-containing RNA (TERRA) is a class of long noncoding RNAs (lncRNAs) that are transcribed from chromosome ends 2,3 ; these RNAs in turn regulate telomeric chromatin structure and telomere maintenance through the telomere-extending enzyme telomerase 4-6 and homology-directed DNA repair 7,8. The mechanisms by which TERRA is recruited to chromosome ends remain poorly defined. Here we develop a reporter system with which to dissect the underlying mechanisms, and show that the UUAGGG repeats of TERRA are both necessary and sufficient to target TERRA to chromosome ends. TERRA preferentially associates with short telomeres through the formation of telomeric DNA-RNA hybrid (R-loop) structures that can form in trans. Telomere association and R-loop formation trigger telomere fragility and are promoted by the recombinase RAD51 and its interacting partner BRCA2, but counteracted by the RNA-surveillance factors RNaseH1 and TRF1. RAD51 physically interacts with TERRA and catalyses R-loop formation with TERRA in vitro, suggesting a direct involvement of this DNA recombinase in the recruitment of TERRA by strand invasion. Together, our findings reveal a RAD51-dependent pathway that governs TERRA-mediated R-loop formation after transcription, providing a mechanism for the recruitment of lncRNAs to new loci in trans. TERRA is transcribed from numerous chromosome ends, and comprises both subtelomeric sequences and telomeric repeats. More than 50% of TERRA is associated with chromatin 9. To investigate how TERRA is recruited to or retained at telomeres, we generated a plasmid encoding 24 copies of the stem-loop of phage PP7 (ref. 10) under the control of the tetracycline-inducible (TET) promoter, followed by 90 TTAGGG repeats (Fig. 1a). To generate full-length TERRA transcripts, we also cloned the human chromosome Xq and 15q subtelomeric regions containing the TERRA start sites between the PP7 stem-loops and the TTAGGG repeats. The constructs were then transiently transfected into HeLa clones that were constitutively expressing the PP7 coat protein fused to GFP (PCP-GFP) and a nuclear-localization signal. PCP-GFP exhibited a diffuse signal in the nucleus but formed nuclear foci upon expression of the PP7 stem-loops, which are bound by PCP and can gather up to 48 PCP-GFP molecules per RNA. These foci did not co-localize with telomeres (Fig. 1b). The fusion of the subtelomeric region of 15q or Xq TERRA to the stem-loops did not promote substantial trafficking of the PP7 foci to telomeres. However, when the telomeric TTAGGG repeats were fused downstream of PP7, co-localization with telomeres occurred, as analysed by conventional and confocal imaging (Fig. 1b), indicating that the 5′-UUAGGG-3′ repeats of TERRA drive telomere association. The full-length PP7-tagged 15q and Xq chimaeric TERRA also showed marked co-localization with telomeres (Fig. 1b and Extended Data Fig. 1a). Therefore, chimaeric TERRAs that originated from a plasmid were directed to telomeres in trans. To eliminate possible confounding effects due to the high plasmid copy number or increased levels of transgenic TERRA, we used CRISPR-Cas9 technology to integrate the chimaeric TERRA constructs into the genome at the adeno-associated-virus integration site 1 (AAVS1) on chromosome 19, which represents a safe harbour for transgene expression 11 (Extended Data Fig. 1b). Following isolation of clones, we confirmed monoallelic site-specific integration of the full constructs by polymerase chain reaction (PCR) and sequencing. These TERRA expression levels were lower than the levels of expression from plasmids, giving one to three foci-indicative of displacement from the transcription site. But, similar to the results obtained upon transient transfection, the PP7 loops formed nuclear foci, and only when fused to 5′-UUAGGG-3′ repeats did the chimaeric RNAs co-localize with telomeres (Extended Data Fig. 1c, d). Shorter telomeres recruit more TERRA In Saccharomyces cerevisiae and Schizosaccharomyces pombe, short telomeres recruit more TERRA, possibly to facilitate telomere maintenance through recombination or telomerase recruitment 5,6,8. To explore the putative roles of telomere length in TERRA recruitment
The proper repair of deleterious DNA lesions such as double strand breaks prevents genomic instab... more The proper repair of deleterious DNA lesions such as double strand breaks prevents genomic instability and carcinogenesis. In yeast, the Rad52 protein mediates DSB repair via homologous recombination. In mammalian cells, despite the presence of the RAD52 protein, the tumour suppressor protein BRCA2 acts as the predominant mediator during homologous recombination. For decades, it has been believed that the RAD52 protein played only a back-up role in the repair of DSBs performing an error-prone single strand annealing (SSA). Recent studies have identified several new functions of the RAD52 protein and have drawn attention to its important role in genome maintenance. Here, we show that RAD52 activities are enhanced by interacting with a small and highly acidic protein called DSS1. Binding of DSS1 to RAD52 changes the RAD52 oligomeric conformation, modulates its DNA binding properties, stimulates SSA activity and promotes strand invasion. Our work introduces for the first time RAD52 as ...
CDK12 is a kinase associated with elongating RNA polymerase II (RNAPII) and is frequently mutated... more CDK12 is a kinase associated with elongating RNA polymerase II (RNAPII) and is frequently mutated in cancer. CDK12 depletion reduces the expression of homologous recombination (HR) DNA repair genes, but comprehensive insight into its target genes and cellular processes is lacking. We use a chemical genetic approach to inhibit analog-sensitive CDK12, and find that CDK12 kinase activity is required for transcription of core DNA replication genes and thus for G1/S progression. RNA-seq and ChIP-seq reveal that CDK12 inhibition triggers an RNAPII processivity defect characterized by a loss of mapped reads from 3 0 ends of predominantly long, poly(A)-signal-rich genes. CDK12 inhibition does not globally reduce levels of RNAPII-Ser2 phosphorylation. However, individual CDK12-dependent genes show a shift of P-Ser2 peaks into the gene body approximately to the positions where RNAPII occupancy and transcription were lost. Thus, CDK12 catalytic activity represents a novel link between regulation of transcription and cell cycle progression. We propose that DNA replication and HR DNA repair defects as a consequence of CDK12 inactivation underlie the genome instability phenotype observed in many cancers.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 2016
Understanding the molecular mechanism of aging could have enormous medical implications. Despite ... more Understanding the molecular mechanism of aging could have enormous medical implications. Despite a century of research, however, there is no universally accepted theory regarding the molecular basis of aging. On the other hand, there is plentiful evidence suggesting that DNA constitutes the central molecule in this process. Here, we review the roles of chromatin structure, DNA damage, and shortening of telomeres in aging and propose a hypothesis for how their interplay leads to aging phenotypes.
Homologous recombination (HR) maintains genome stability by repairing DNA double-strand breaks an... more Homologous recombination (HR) maintains genome stability by repairing DNA double-strand breaks and gaps and restarting replication forks. It is an error-free pathway that uses a homologous sequence in the genome to copy the damaged genetic information. In the present chapter, we will discuss in detail the mechanism by which HR operates to maintain genome stability as revealed by studies predominantly performed in Saccharomyces cerevisiae. We will then discuss the similarities and dissimilarities between yeast and humans while emphasizing the importance of HR in suppressing carcinogenesis and as a potential therapeutic target.
A variety of DNA lesions, secondary DNA structures or topological stress within the DNA template ... more A variety of DNA lesions, secondary DNA structures or topological stress within the DNA template may lead to stalling of the replication fork. Recovery of such forks is essential for the maintenance of genomic stability. The structure-specific endonuclease Mus81-Mms4 has been implicated in processing DNA intermediates that arise from collapsed forks and homologous recombination. According to previous genetic studies, the Srs2 helicase may play a role in the repair of double-strand breaks and ssDNA gaps together with Mus81-Mms4. In this study, we show that the Srs2 and Mus81-Mms4 proteins physically interact in vitro and in vivo and we map the interaction domains within the Srs2 and Mus81 proteins. Further, we show that Srs2 plays a dual role in the stimulation of the Mus81-Mms4 nuclease activity on a variety of DNA substrates. First, Srs2 directly stimulates Mus81-Mms4 nuclease activity independent of its helicase activity. Second, Srs2 removes Rad51 from DNA to allow access of Mus81-Mms4 to cleave DNA. Concomitantly, Mus81-Mms4 inhibits the helicase activity of Srs2. Taken together, our data point to a coordinated role of Mus81-Mms4 and Srs2 in processing of recombination as well as replication intermediates.
Rad54 is an ATP-driven translocase involved in the genome maintenance pathway of homologous recom... more Rad54 is an ATP-driven translocase involved in the genome maintenance pathway of homologous recombination (HR). Although its activity has been implicated in several steps of HR, its exact role(s) at each step are still not fully understood. We have identified a new interaction between Rad54 and the replicative DNA clamp, proliferating cell nuclear antigen (PCNA). This interaction was only mildly weakened by the mutation of two key hydrophobic residues in the highlyconserved PCNA interaction motif (PIP-box) of Rad54 (Rad54-AA). Intriguingly, the rad54-AA mutant cells displayed sensitivity to DNA damage and showed HR defects similar to the null mutant, despite retaining its ability to interact with HR proteins and to be recruited to HR foci in vivo. We therefore surmised that the PCNA interaction might be impaired in vivo and was unable to promote repair synthesis during HR. Indeed, the Rad54-AA mutant was defective in primer extension at the MAT locus as well as in vitro, but additional biochemical analysis revealed that this mutant also had diminished ATPase activity and an inability to promote D-loop formation. Further mutational analysis of the putative PIP-box uncovered that other phenotypically relevant mutants in this domain also resulted in a loss of ATPase activity. Therefore, we have found that although Rad54 interacts with PCNA, the PIP-box motif likely plays only a minor role in stabilizing the PCNA interaction, and rather, this conserved domain is probably an extension of the ATPase domain III.
The biological functions of the small ubiquitin-related Clare Hall Laboratories modifier SUMO app... more The biological functions of the small ubiquitin-related Clare Hall Laboratories modifier SUMO appear to be even more diverse, rang-Blanche Lane ing from nuclear transport to signal transduction, tran-South Mimms scription, and genome stability (Johnson, 2004). At-Herts EN6 3LD tachment of SUMO can induce a relocalization of the United Kingdom target protein within the cell, a conformational change, 2 Max Planck Institute for Terrestrial Microbiology or an alteration of protein-protein interactions, but Karl-von-Frisch-Strasse often its mode of action remains poorly understood. D-35043 Marburg Despite a similar conjugation mechanism, ubiquitin and Germany SUMO generally direct their targets to very different 3 Department of Molecular Biophysics and fates (Gill, 2004). This notion becomes most obvious in Biochemistry situations where the two modifiers compete for the School of Medicine same attachment site on a common substrate protein, Yale University as first noted in the case of the NF-κB inhibitor IκBα, 333 Cedar Street where sumoylation was found to counteract the proteo-New Haven, Connecticut 06520 lytic effect of ubiquitination (Desterro et al., 1998). In the context of DNA replication and repair, SUMO and ubiquitin jointly affect a key signal integrator at the Summary replication fork, PCNA. Besides its function as a sliding clamp that ensures the processivity of replicative DNA Posttranslational modification of proliferating cell polymerases, PCNA serves as a binding platform for nuclear antigen (PCNA), an essential processivity various enzymes involved in DNA repair, chromatin asclamp for DNA polymerases, by ubiquitin and SUMO sembly, and cell cycle control (Jónsson and Hübscher, contributes to the coordination of DNA replication, 1997). In response to DNA-damaging agents, PCNA is damage tolerance, and mutagenesis. Whereas ubiqubiquitinated at a highly conserved lysine (K) residue, uitination in response to DNA damage promotes the K164 (Hoege et al., 2002). In the yeast Saccharomyces bypass of replication-blocking lesions, sumoylation cerevisiae, the same lysine is modified by SUMO during during S phase is damage independent. As both mod-S phase, independent of DNA damage. A second, less ifiers target the same site on PCNA, an antagonistic conserved lysine, K127, is used as a secondary attachaction of SUMO on ubiquitin-dependent DNA damage ment site for SUMO. Ubiquitination of PCNA is critical tolerance has been proposed. We now present evifor the cell's survival in the presence of DNA damage, dence that the apparent negative effect of SUMO on as it facilitates the bypass of replication-blocking lesion bypass is not due to competition with ubiquitilesions. By modification of PCNA, the ubiquitin system nation but is rather mediated by the helicase Srs2p, in fact promotes DNA damage tolerance in two distinct which affects genome stability by suppressing unmodes: whereas monoubiquitination triggers transscheduled homologous recombination. We show that lesion synthesis and damage-induced mutagenesis by Srs2p physically interacts with sumoylated PCNA, error-prone polymerases (Kannouche et al., 2004; which contributes to the recruitment of the helicase Stelter and Ulrich, 2003; Watanabe et al., 2004), polyto replication forks. Our findings suggest a mechaubiquitination activates an error-free damage avoidnism by which SUMO and ubiquitin cooperatively ance pathway that is believed to utilize the genetic incontrol the choice of pathway for the processing of formation encoded by the undamaged sister chromatid DNA lesions during replication. for lesion bypass (Hoege et al., 2002). In the absence of DNA damage, the error-prone polymerase ζ also
The budding yeast Srs2 protein possesses 3 to 5 DNA helicase activity and channels untimely recom... more The budding yeast Srs2 protein possesses 3 to 5 DNA helicase activity and channels untimely recombination to post-replication repair by removing Rad51 from ssDNA. However, it also promotes recombination via a synthesis-dependent strand-annealing pathway (SDSA). Furthermore, at the replication fork, Srs2 is required for fork progression and prevents the instability of trinucleotide repeats. To better understand the multiple roles of the Srs2 helicase during these processes, we analysed the ability of Srs2 to bind and unwind various DNA substrates that mimic structures present during DNA replication and recombination. While leading or lagging strands were efficiently unwound, the presence of ssDNA binding protein RPA presented an obstacle for Srs2 translocation. We also tested the preferred directionality of unwinding of various substrates and studied the effect of Rad51 and Mre11 proteins on Srs2 helicase activity. These biochemical results help us understand the possible role of Srs2 in the processing of stalled or blocked replication forks as a part of post-replication repair as well as homologous recombination (HR).
Homologous recombination plays a key role in the maintenance of genome integrity, especially duri... more Homologous recombination plays a key role in the maintenance of genome integrity, especially during DNA replication and the repair of double-stranded DNA breaks (DSBs). Just a single un-repaired break can lead to aneuploidy, genetic aberrations or cell death. DSBs are caused by a vast number of both endogenous and exogenous agents including genotoxic chemicals or ionizing radiation, as well as through replication of a damaged template DNA or the replication fork collapse. It is essential for cell survival to recognise and process DSBs as well as other toxic intermediates and launch most appropriate repair mechanism. Many helicases have been implicated to play role in these processes, however their detail roles, specificities and co-operativity in the complex protein-protein interaction networks remain unclear. In this review we summarize the current knowledge about Saccharomyces cerevisiae helicase Srs2 and its effect on multiple DNA metabolic processes that generally affect genome stability. It would appear that Srs2 functions as an "Odd-Job Man" in these processes to make sure that the jobs proceed when and where they are needed.
DNA damage tolerance (DDT) and homologous recombination (HR) stabilize replication forks (RFs). R... more DNA damage tolerance (DDT) and homologous recombination (HR) stabilize replication forks (RFs). RAD18/UBC13/three prime repair exonuclease 2 (TREX2)-mediated proliferating cell nuclear antigen (PCNA) ubiquitination is central to DDT, an error-prone lesion bypass pathway. RAD51 is the recombinase for HR. The RAD51 K133A mutation increased spontaneous mutations and stress-induced RF stalls and nascent strand degradation. Here, we report in RAD51 K133A cells that this phenotype is reduced by expressing a TREX2 H188A mutation that
Cdk1 kinase phosphorylates budding yeast Srs2, a member of UvrD protein family that displays both... more Cdk1 kinase phosphorylates budding yeast Srs2, a member of UvrD protein family that displays both DNA translocation and DNA unwinding activities in vitro. Srs2 prevents homologous recombination by dismantling Rad51 filaments and it is also required for double strand break repair. Here we examine the biological significance of Cdk1-dependent phosphorylation of Srs2 using mutants that constitutively express the phosphorylated or unphosphorylated isoforms. We found that Cdk1 targets Srs2 to repair double strand break (DSB) after strand invasion. Srs2 phosphorylation is required to complete synthesis-dependent strand annealing pathway, likely controlling the disassembly of the D-loop intermediate. Cdk1 phosphorylation, indeed, controls the turnover of Srs2 protein at the invading strand, while it is not required for that of Rad51. Further analysis on the recombination phenotypes of the srs2 phospho-mutants indicated that Srs2 phosphorylation is not indeed essential for the removal of to...
Homologous recombination (HR) is a process widespread in nature and essential to maintain the int... more Homologous recombination (HR) is a process widespread in nature and essential to maintain the integrity of the genome via repair of DNA double-strand breaks (DSBs). Rad52 is a DNA-binding protein which plays a key role in HR. It mediates the exchange of the recombination factor RPA associated to single-stranded DNA (ssDNA) by Rad51, resulting in the assembly of the presynaptic filament. Recently, it has been reported that upon DNA damage Rad52 is modified by the small ubiquitin-like modifier (SUMO) protein which shelters Rad52 against proteosomal degradation. As the major SUMOylation sites of Rad52, three lysines (K43, K44, K253) have been identified. The effect of Rad52 DNA binding and interaction with its partners was tested to clarify the regulatory mechanism of Rad52 SUMOylation.
Rothmund-Thomsonův syndrom (RTS) je autozomalni recesivni onemocněni. Typickým klinickým přiznake... more Rothmund-Thomsonův syndrom (RTS) je autozomalni recesivni onemocněni. Typickým klinickým přiznakem RTS je cervena vyražka, znama jako poikiloderma. Pacienti navic trpi ocnim zakalem, kosternimi defekty a vyssi nachylnosti k rakovině kůže a kosti. Mutace v genu RECQ4, ktere byly zjistěny u pacientů s RTS, vedou k produkci nefunkcnich verzi proteinu RecQ4. Domeny proteinu RecQ4 jsou velmi podobne jako u ostatnich clenů RecQ helikaz, avsak jeho přesna funkce je dosud neznama. Nicmeně, protein RecQ4 se podili na udržovani stability geneticke informace, hraje významnou roli při replici a opravě DNA.
Extracellular pH has been assumed to play little if any role in how bacteria respond to antibioti... more Extracellular pH has been assumed to play little if any role in how bacteria respond to antibiotics and antibiotic resistance development. Here, we show that the intracellular pH of Escherichia coli equilibrates to the environmental pH following antibiotic treatment. We demonstrate that this allows the environmental pH to influence the transcription of various DNA damage response genes and physiological processes such as filamentation. Using purified RecA and a known pH-sensitive mutant variant RecA K250R we show how pH can affect the biochemical activity of a protein central to control of the bacterial DNA damage response system. Finally, two different mutagenesis assays indicate that environmental pH affects antibiotic resistance development. Specifically, at environmental pH's greater than six we find that mutagenesis plays a significant role in producing antibiotic resistant mutants. At pH's less than or equal to 6 the genome appears more stable but extensive filamentati...
Dna2 is an essential nuclease-helicase that acts in several distinct DNA metabolic pathways inclu... more Dna2 is an essential nuclease-helicase that acts in several distinct DNA metabolic pathways including DNA replication and recombination. To balance these functions and prevent unscheduled DNA degradation, Dna2 activities must be regulated. Here we show that Saccharomyces cerevisiae Dna2 function is controlled by sumoylation. We map the sumoylation sites to the N-terminal regulatory domain of Dna2 and show that in vitro sumoylation of recombinant Dna2 impairs its nuclease but not helicase activity. In cells, the total levels of the non-sumoylatable Dna2 variant are elevated. However, non-sumoylatable Dna2 shows impaired nuclear localization and reduced recruitment to foci upon DNA damage. Nonsumoylatable Dna2 reduces the rate of DNA end resection, as well as impedes cell growth and cell cycle progression through S phase. Taken together, these findings show that in addition to Dna2 phosphorylation described previously, Dna2 sumoylation is required for the homeostasis of the Dna2 protein function to promote genome stability.
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