The role of the DNA damage checkpoint in regulation of translesion DNA synthesis

Living organisms are continuously under threat from a vast array of DNA-damaging agents, which impact genome DNA. DNA replication machinery stalls at damaged template DNA. The stalled replication fork is restarted via bypass replication by translesion DNA-synthesis polymerases, including the Y-family polymerases Polη, Polι, and Polκ, which possess the ability to incorporate nucleotides opposite the damaged template. To investigate the division of labor among these polymerases in vivo, we generated POLη−/−, POLι−/−, POLκ−/−, double knockout (KO), and triple knockout (TKO) mutants in all combinations from human TK6 cells. TKO cells exhibited a hypersensitivity to ultraviolet (UV), cisplatin (CDDP), and methyl methanesulfonate (MMS), confirming the pivotal role played by these polymerases in bypass replication of damaged template DNA. POLη−/− cells, but not POLι−/− or POLκ−/− cells, showed a strong sensitivity to UV and CDDP, while TKO cells showed a slightly higher sensitivity to UV and CDDP than did POLη−/− cells. On the other hand, TKO cells, but not all single KO cells, exhibited a significantly higher sensitivity to MMS than did wild-type cells. Consistently, DNA-fiber assay revealed that Polη plays a crucial role in bypassing lesions caused by UV-mimetic agent 4-nitroquinoline-1-oxide and CDDP, while all three polymerases play complementary roles in bypassing MMS-induced damage. Our findings indicate that the three Y-family polymerases play distinctly different roles in bypass replication, according to the type of DNA damage generated on the template strand.

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Investigating the role of the E. coli β‐sliding clamp in DNA polymerase V‐dependent translesion DNA synthesis

The FASEB Journal ◽

10.1096/fasebj.20.5.a909-c ◽

2006 ◽

Vol 20 (5) ◽

Author(s):

Mark David Sutton ◽

Laurie Sanders ◽

Sarah Ponticelli ◽

Jill Duzen ◽

Robert Maul ◽

...

Keyword(s):

Dna Synthesis ◽

Dna Polymerase ◽

Translesion Dna Synthesis ◽

E Coli ◽

Sliding Clamp ◽

Dna Polymerase V ◽

Translesion Dna

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The role of HERC2 and RNF8 ubiquitin E3 ligases in the promotion of translesion DNA synthesis in the chicken DT40 cell line

DNA Repair ◽

10.1016/j.dnarep.2016.02.002 ◽

2016 ◽

Vol 40 ◽

pp. 67-76 ◽

Cited By ~ 12

Author(s):

Mohiuddin ◽

Shunsuke Kobayashi ◽

Islam Shamima Keka ◽

Guillaume Guilbaud ◽

Julian Sale ◽

...

Keyword(s):

Dna Synthesis ◽

Cell Line ◽

E3 Ligases ◽

Translesion Dna Synthesis ◽

Dt40 Cell ◽

Translesion Dna ◽

Chicken Dt40 Cell

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The Role of Specific Amino Acid Residues in the Active Site ofEscherichia coliDNA Polymerase I on Translesion DNA Synthesis Across from and Past anN-2-Aminofluorene Adduct†

Biochemistry ◽

10.1021/bi061324o ◽

2007 ◽

Vol 46 (10) ◽

pp. 2599-2607 ◽

Cited By ~ 3

Author(s):

Samer Lone ◽

Louis J. Romano

Keyword(s):

Amino Acid ◽

Dna Synthesis ◽

Active Site ◽

Amino Acid Residues ◽

Specific Amino Acid ◽

Translesion Dna Synthesis ◽

Translesion Dna ◽

Polymerase I

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PDIP38/PolDIP2 controls the DNA damage tolerance pathways by increasing the relative usage of translesion DNA synthesis over template switching

PLoS ONE ◽

10.1371/journal.pone.0213383 ◽

2019 ◽

Vol 14 (3) ◽

pp. e0213383 ◽

Cited By ~ 3

Author(s):

Masataka Tsuda ◽

Saki Ogawa ◽

Masato Ooka ◽

Kaori Kobayashi ◽

Kouji Hirota ◽

...

Keyword(s):

Dna Damage ◽

Dna Synthesis ◽

Damage Tolerance ◽

Template Switching ◽

Dna Damage Tolerance ◽

Translesion Dna Synthesis ◽

Translesion Dna

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Accessory proteins assist exonuclease-deficient bacteriophage T4 DNA polymerase in replicating past an abasic site

Biochemical Journal ◽

10.1042/bj20060898 ◽

2007 ◽

Vol 402 (2) ◽

pp. 321-329 ◽

Cited By ~ 4

Author(s):

Giuseppina Blanca ◽

Emmanuelle Delagoutte ◽

Nicolas Tanguy le gac ◽

Neil P. Johnson ◽

Giuseppe Baldacci ◽

...

Keyword(s):

Dna Damage ◽

Dna Synthesis ◽

Dna Polymerase ◽

Bacteriophage T4 ◽

Dna Polymerases ◽

Accessory Proteins ◽

Clamp Loader ◽

Abasic Site ◽

Translesion Dna Synthesis ◽

Translesion Dna

Replicative DNA polymerases, such as T4 polymerase, possess both elongation and 3′–5′ exonuclease proofreading catalytic activities. They arrest at the base preceding DNA damage on the coding DNA strand and specialized DNA polymerases have evolved to replicate across the lesion by a process known as TLS (translesion DNA synthesis). TLS is considered to take place in two steps that often require different enzymes, insertion of a nucleotide opposite the damaged template base followed by extension from the inserted nucleotide. We and others have observed that inactivation of the 3′–5′ exonuclease function of T4 polymerase enables TLS across a single site-specific abasic [AP (apurinic/apyrimidinic)] lesion. In the present study we report a role for auxiliary replicative factors in this reaction. When replication is performed with a large excess of DNA template over DNA polymerase in the absence of auxiliary factors, the exo− polymerase (T4 DNA polymerase deficient in the 3′–5′ exonuclease activity) inserts one nucleotide opposite the AP site but does not extend past the lesion. Addition of the clamp processivity factor and the clamp loader complex restores primer extension across an AP lesion on a circular AP-containing DNA substrate by the exo− polymerase, but has no effect on the wild-type enzyme. Hence T4 DNA polymerase exhibits a variety of responses to DNA damage. It can behave as a replicative polymerase or (in the absence of proofreading activity) as a specialized DNA polymerase and carry out TLS. As a specialized polymerase it can function either as an inserter or (with the help of accessory proteins) as an extender. The capacity to separate these distinct functions in a single DNA polymerase provides insight into the biochemical requirements for translesion DNA synthesis.

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Homologous Recombination and Translesion DNA Synthesis Play Critical Roles on Tolerating DNA Damage Caused by Trace Levels of Hexavalent Chromium

PLoS ONE ◽

10.1371/journal.pone.0167503 ◽

2016 ◽

Vol 11 (12) ◽

pp. e0167503 ◽

Cited By ~ 5

Author(s):

Xu Tian ◽

Keyur Patel ◽

John R. Ridpath ◽

Youjun Chen ◽

Yi-Hui Zhou ◽

...

Keyword(s):

Dna Damage ◽

Homologous Recombination ◽

Dna Synthesis ◽

Hexavalent Chromium ◽

Translesion Dna Synthesis ◽

Translesion Dna

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Identification of novel DNA-damage tolerance genes reveals regulation of translesion DNA synthesis by nucleophosmin

Nature Communications ◽

10.1038/ncomms6437 ◽

2014 ◽

Vol 5 (1) ◽

Cited By ~ 27

Author(s):

Omer Ziv ◽

Amit Zeisel ◽

Nataly Mirlas-Neisberg ◽

Umakanta Swain ◽

Reinat Nevo ◽

...

Keyword(s):

Dna Damage ◽

Dna Synthesis ◽

Damage Tolerance ◽

Dna Damage Tolerance ◽

Translesion Dna Synthesis ◽

Translesion Dna

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Characterization of human translesion DNA synthesis across a UV-induced DNA lesion

eLife ◽

10.7554/elife.19788 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 26

Author(s):

Mark Hedglin ◽

Binod Pandey ◽

Stephen J Benkovic

Keyword(s):

Dna Synthesis ◽

S Phase ◽

Post Translational Modification ◽

Translesion Dna Synthesis ◽

Quantitative Evidence ◽

Dna Lesion ◽

Translesion Dna ◽

Lagging Strand

Translesion DNA synthesis (TLS) during S-phase uses specialized TLS DNA polymerases to replicate a DNA lesion, allowing stringent DNA synthesis to resume beyond the offending damage. Human TLS involves the conjugation of ubiquitin to PCNA clamps encircling damaged DNA and the role of this post-translational modification is under scrutiny. A widely-accepted model purports that ubiquitinated PCNA recruits TLS polymerases such as pol η to sites of DNA damage where they may also displace a blocked replicative polymerase. We provide extensive quantitative evidence that the binding of pol η to PCNA and the ensuing TLS are both independent of PCNA ubiquitination. Rather, the unique properties of pols η and δ are attuned to promote an efficient and passive exchange of polymerases during TLS on the lagging strand.

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