Unscheduled DNA Synthesis: The Clinical and Functional Assay for Global Genomic DNA Nucleotide Excision Repair

Nucleotide excision repair is the sole mechanism for removing the major UV photoproducts from genomic DNA in human cells. In vitro with human cell-free extract or purified excision repair factors, the damage is removed from naked DNA or nucleosomes in the form of 24- to 32-nucleotide-long oligomers (nominal 30-mer) by dual incisions. Whether the DNA damage is removed from chromatin in vivo in a similar manner and what the fate of the excised oligomer was has not been known previously. Here, we demonstrate that dual incisions occur in vivo identical to the in vitro reaction. Further, we show that transcription-coupled repair, which operates in the absence of the XPC protein, also generates the nominal 30-mer in UV-irradiated XP-C mutant cells. Finally, we report that the excised 30-mer is released from the chromatin in complex with the repair factors TFIIH and XPG. Taken together, our results show the congruence of in vivo and in vitro data on nucleotide excision repair in humans.

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Nucleotide Excision Repair or Polymerase V-Mediated Lesion Bypass Can Act To Restore UV-Arrested Replication Forks in Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.187.20.6953-6961.2005 ◽

2005 ◽

Vol 187 (20) ◽

pp. 6953-6961 ◽

Cited By ~ 35

Author(s):

Charmain T. Courcelle ◽

Jerilyn J. Belle ◽

Justin Courcelle

Keyword(s):

Dna Synthesis ◽

Nucleotide Excision Repair ◽

Excision Repair ◽

Translesion Synthesis ◽

Replication Forks ◽

Repair Capacity ◽

Translesion Dna Synthesis ◽

Pol V ◽

Nucleotide Excision ◽

Translesion Dna

ABSTRACT Nucleotide excision repair and translesion DNA synthesis are two processes that operate at arrested replication forks to reduce the frequency of recombination and promote cell survival following UV-induced DNA damage. While nucleotide excision repair is generally considered to be error free, translesion synthesis can result in mutations, making it important to identify the order and conditions that determine when each process is recruited to the arrested fork. We show here that at early times following UV irradiation, the recovery of DNA synthesis occurs through nucleotide excision repair of the lesion. In the absence of repair or when the repair capacity of the cell has been exceeded, translesion synthesis by polymerase V (Pol V) allows DNA synthesis to resume and is required to protect the arrested replication fork from degradation. Pol II and Pol IV do not contribute detectably to survival, mutagenesis, or restoration of DNA synthesis, suggesting that, in vivo, these polymerases are not functionally redundant with Pol V at UV-induced lesions. We discuss a model in which cells first use DNA repair to process replication-arresting UV lesions before resorting to mutagenic pathways such as translesion DNA synthesis to bypass these impediments to replication progression.

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Nucleotide Excision Repair DNA Synthesis by DNA Polymerase .epsilon. in the Presence of PCNA, RFC, and RPA

Biochemistry ◽

10.1021/bi00015a012 ◽

1995 ◽

Vol 34 (15) ◽

pp. 5011-5017 ◽

Cited By ~ 160

Author(s):

Mahmud K. K. Shivji ◽

Vladimir N. Podust ◽

Ulrich Huebscher ◽

Richard D. Wood

Keyword(s):

Dna Synthesis ◽

Nucleotide Excision Repair ◽

Dna Polymerase ◽

Excision Repair ◽

Dna Polymerase Epsilon ◽

Nucleotide Excision ◽

Polymerase Epsilon

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UvrD Participation in Nucleotide Excision Repair Is Required for the Recovery of DNA Synthesis following UV-Induced Damage inEscherichia coli

Journal of Nucleic Acids ◽

10.1155/2012/271453 ◽

2012 ◽

Vol 2012 ◽

pp. 1-9 ◽

Cited By ~ 7

Author(s):

Kelley N. Newton ◽

Charmain T. Courcelle ◽

Justin Courcelle

Keyword(s):

Dna Damage ◽

Dna Synthesis ◽

Nucleotide Excision Repair ◽

Excision Repair ◽

Dna Helicase ◽

Replication Forks ◽

Nucleotide Excision ◽

Fork Regression

UvrD is a DNA helicase that participates in nucleotide excision repair and several replication-associated processes, including methyl-directed mismatch repair and recombination. UvrD is capable of displacing oligonucleotides from synthetic forked DNA structuresin vitroand is essential for viability in the absence of Rep, a helicase associated with processing replication forks. These observations have led others to propose that UvrD may promote fork regression and facilitate resetting of the replication fork following arrest. However, the molecular activity of UvrD at replication forksin vivohas not been directly examined. In this study, we characterized the role UvrD has in processing and restoring replication forks following arrest by UV-induced DNA damage. We show that UvrD is required for DNA synthesis to recover. However, in the absence of UvrD, the displacement and partial degradation of the nascent DNA at the arrested fork occur normally. In addition, damage-induced replication intermediates persist and accumulate inuvrDmutants in a manner that is similar to that observed in other nucleotide excision repair mutants. These data indicate that, following arrest by DNA damage, UvrD is not required to catalyze fork regressionin vivoand suggest that the failure ofuvrDmutants to restore DNA synthesis following UV-induced arrest relates to its role in nucleotide excision repair.

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