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    DNA Damage Repair Mechanisms

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    prianna
    DNA repair is essential for cells to correct errors that occur during DNA replication and from damage by environmental factors. There are two major pathways for repairing DNA damage - base excision repair removes individual nucleotides while nucleotide excision repair removes larger sections of DNA containing damage. Double strand breaks pose a serious risk and are repaired through either non-homologous end joining, which quickly joins the ends, or homologous recombination, which uses a sister chromatid as a template for accurate repair. Specialized repair mechanisms like transcription-coupled repair and DNA damage checkpoints also help preserve genome integrity.

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    DNA Damage Repair Mechanisms

    Uploaded by

    prianna
    130 views40 pages
    DNA repair is essential for cells to correct errors that occur during DNA replication and from damage by environmental factors. There are two major pathways for repairing DNA damage - base excision repair removes individual nucleotides while nucleotide excision repair removes larger sections of DNA containing damage. Double strand breaks pose a serious risk and are repaired through either non-homologous end joining, which quickly joins the ends, or homologous recombination, which uses a sister chromatid as a template for accurate repair. Specialized repair mechanisms like transcription-coupled repair and DNA damage checkpoints also help preserve genome integrity.

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    Slides on DNA damage repair mechanisms from UCLA MCDB.

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    DNA repair is essential for cells to correct errors that occur during DNA replication and from damage by environmental factors. There are two major pathways for repairing DNA damage - base excision repair removes individual nucleotides while nucleotide excision repair removes larger sections of DNA containing damage. Double strand breaks pose a serious risk and are repaired through either non-homologous end joining, which quickly joins the ends, or homologous recombination, which uses a sister chromatid as a template for accurate repair. Specialized repair mechanisms like transcription-coupled repair and DNA damage checkpoints also help preserve genome integrity.

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    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
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    130 views40 pages

    DNA Damage Repair Mechanisms

    Uploaded by

    prianna
    DNA repair is essential for cells to correct errors that occur during DNA replication and from damage by environmental factors. There are two major pathways for repairing DNA damage - base excision repair removes individual nucleotides while nucleotide excision repair removes larger sections of DNA containing damage. Double strand breaks pose a serious risk and are repaired through either non-homologous end joining, which quickly joins the ends, or homologous recombination, which uses a sister chromatid as a template for accurate repair. Specialized repair mechanisms like transcription-coupled repair and DNA damage checkpoints also help preserve genome integrity.

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    of 40

    Lecture #5

    DNA Damage and mechanisms of repair


    MBOC (old) Page 267-285 MBOC (new) Page 295-311

    Concepts
    ! Factors that can induce DNA Damage ! Describe repair mechanisms for the following errors
    ! Single nucleotide errors ! Small regions of mismatch ! Double strand breaks

    DNA Replication has Proofreading mechanisms


    ! DNA polymerase has two proofreading mechanisms
    ! Correct nucleotide has high affinity for the moving polymerase ! Conformational change in DNA polymerase is required after nucleotide binding but before covalent addition check DNA strand

    ! Exonucleolytic proof-reading activity


    ! Only adds to the 3 -OH of a primer strand ! Mismatched nucleotides do not make a correct basepair, thus disrupting the primer

    DNA editing mode of DNA Polymerase

    DNA editing mode of DNA pol

    DNA polymerase

    What happens in catastrophic cases of DNA damage?


    ! If a cells DNA is heavily damaged the classic DNA repair pathways can not operate ! Replicative DNA polymerase stalls when it encounters DNA damage in catastrophic emergencies
    ! less accurate back-up DNA polymerases are used ! Human cells have >10 such polymerases ! Some recognize specific types of DNA damage ! Others make good guesses

    ! Lack exonuclease proof reading activity

    Why is DNA repair so important?

    What creates and/or propagates DNA damage?


    ! UV radiation (sun burn) ! Ionizing radiation (X-ray machines) ! Chemicals (ENU)
    Causes irreparable damage to male germline cells, studied in labs.

    ! Errors in DNA repair mechanisms

    Release of the radioactive isotope I131

    Source: US AEC 1955

    Above ground atomic test site - Nevada

    "The greatest irony of our atmospheric nuclear testing program is that the only victims of United States nuclear arms since World War II have been our own people." -Congressional investigation team member

    Ken Case Atomic cowboy

    Syndromes in Humans caused by defects in DNA repair machinery

    Repairing a single nucleotide error

    Some types of DNA damage occurs spontaneously in cells


    ! DNA in human cells lose ~ 5,000 purine bases (A, G) every day
    ! N-Glycosyl linkage to deoxyribose hydrolyses
    ! DEPURINATION

    ! Removal of amine groups from bases


    ! DEAMINATION
    ! Every 100 bases per cell every day
    A,G,C but not T Hydrolysis of amine. Methylated cytosine deaminates to thymine

    Example of Depurination

    Result is absent base but an intact sugar phosphate backbone


    Text

    Example of Deamination

    This hydrolysis occurs ~100 bases per day

    Special circumstance of methylated C residues

    ~ 3% of C residues are methylated in vertebrates Deamination of C residues results in generation of

    Pyrimidine dimers
    ! Usually occurs in cells exposed to UV irradiation ! These form between two neighboring pyrimidines ! The most common are thymidine dimers

    Method by which chemical modifications produce mutations


    Cells derived from mutated strand are abnormal

    Normal cells derived from complementary strand

    Base Excision Repair


    ! Uses enzymes called DNA glycosylases ! Recognizes altered bases in DNA and catalyzes its removal from the sugar ! Recognition is based upon a flipping-out mechanism ! The catalyzed base is recognized by an APendonuclease ! DNA polymerase repairs the gap

    DNA Glycosylases detect flipping out

    Base excision repair

    Base gap

    Single nucleotide gap.

    Removing more than one base from a single strand

    Nucleotide Excision Repair


    ! Removes large changes in structure of DNA ! Multienzyme complex scans DNA for distortions ! Cleaves the DNA strand, requires the activity of DNA polymerase and Ligase to cooperate a segment of DNA rather than individual/single bases. in DNA repair Removes Like base excision repair: requires polymerase, ligase.

    Example - Pyrimidine dimers

    Covalent bonds bind pyrimidines together C or T

    Nucleotide Excision Repair

    1. Excision nuclease creates two nicks in the DNA strand.

    Transcription-Coupled Repair
    ! Genomic DNA is constantly under surveillance ! DNA polymerase has inbuilt repair mechanisms during replication ! RNA polymerase will also stall at genetic lesions
    ! interact with coupling proteins ! recruits repair machinery to sites of mismatch

    ! This is important for genes being expressed at the time of DNA damage

    Cockayne s Syndrome
    -! Homozygous recessive disorder -! Children age very quickly (this child is 8 years old) -! Growth retardation, skeletal abnormalities, severe sensitivity to sunlight -! Syndrome is believed to perpetuate because RNA polymerase becomes stalled at sites of DNA damage in important genes

    -! As a result, important genes are not transcribed

    Repair of Double Strand Breaks

    Repair of Double Strand Breaks


    ! This occurs when both DNA strands are cleaved ! Ionizing radiation (I131, replication errors, oxidizing agents cell metabolites ! If not repaired, this can result in loss of chromosomal regions and genes which are not segregated in mitosis ! Two major mechanisms exist to repair double strand breaks
    ! Non-homologous end joining ! Homologous recombination

    Non-Homologous End joining

    Non-Homologous end joining

    Quick and Dirty

    NHEJ is a common repair mechanism used by cells

    DNA Ligase IV
    ! Acts specifically with XRCC1 (X ray repair cross complementing protein) in non homologous end joining (NHEJ) to covalently link two strands of DNA
    Acts specically in non-homologous end joining.

    ! Involves two copies of XRCC1 and DNA ligase IV to bind to DNA with nicks or broken ends
    Complex would involve two each of Ku and XRCC1 proteins and two copies of DNA ligase IV.

    Sister chromatid based repair happens in S or G2 or M.

    Homologous Recombination

    DNA Damage and the Cell Cycle


    BLOCK

    SLOW

    BLOCK

    Rapid localization of repair proteins to double strand breaks


    Dark regions protected from radiation exposure.

    DAPI e
    Regions that uoresce have incorporated BrdU (synthetic base) into DNA, indicating that damage and subsequent repair of damaged bases with BrdU has occured.

    BrdU
    Mre11 is a repair protein. Red banded regions indicate that Mre11 is co-localizing with BrdU.

    Mre11

    Summary
    ! Most damage to DNA can be repaired by one of two major DNA repair pathways ! Double strand breaks pose a larger risk to the cell, and dire consequences for a cell s biology including predisposition to cancer ! Double strand breaks are repaired by a quick and dirty non homologous end joining method or by homologous recombination

    Summary
    ! DNA repair is a fundamental process required in all cells ! Basic mechanisms to repair nucleotide errors utilize DNA glycosylases, DNA polymerase, DNA ligase ! Double strand breaks are repaired by nonhomologous end joining or by homologous recombination

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