POLN, a Nuclear PolA Family DNA Polymerase Homologous to the DNA Cross-link Sensitivity Protein Mus308

Marini, Federica; Kim, Nayun; Schuffert, Anthony; Wood, Richard D.

doi:10.1074/jbc.m305646200

Cited by 105 publications

(122 citation statements)

References 32 publications

(15 reference statements)

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“…TLS polymerases are catalytically less efficient than replicative enzymes and are much more error-prone because of their open and flexible active sites that accommodate large DNA adducts (43, 49 -51) and their lack of intrinsic 3Ј 3 5Ј proofreading activity (41,46,(52)(53)(54)(55). In humans, TLS polymerases include Y family polymerases hPols , , and and Rev1, A family Pol , and B family Pol (41,(43)(44)(45)(46)(47)(48)(49). Polymerase switching is thought to be regulated, at least in part, by posttranslational modifications of the proliferating cell nuclear antigen sliding clamp, which acts as a scaffold for polymerase enzymes (41,(43)(44)(45)(46)(47)(48)(49).…”

Section: Discussionmentioning

confidence: 99%

“…In humans, TLS polymerases include Y family polymerases hPols , , and and Rev1, A family Pol , and B family Pol (41,(43)(44)(45)(46)(47)(48)(49). Polymerase switching is thought to be regulated, at least in part, by posttranslational modifications of the proliferating cell nuclear antigen sliding clamp, which acts as a scaffold for polymerase enzymes (41,(43)(44)(45)(46)(47)(48)(49).…”

Section: Discussionmentioning

confidence: 99%

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Bypass of DNA-Protein Cross-links Conjugated to the 7-Deazaguanine Position of DNA by Translesion Synthesis Polymerases

Wickramaratne

Mukherjee

et al. 2016

Journal of Biological Chemistry

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DNA-protein cross-links (DPCs) are bulky DNA lesions that form both endogenously and following exposure to bis-electrophiles such as common antitumor agents. The structural and biological consequences of DPCs have not been fully elucidated due to the complexity of these adducts. The most common site of DPC formation in DNA following treatment with bis-electrophiles such as nitrogen mustards and cisplatin is the N7 position of guanine, but the resulting conjugates are hydrolytically labile and thus are not suitable for structural and biological studies. In this report, hydrolytically stable structural mimics of N7-guanine-conjugated DPCs were generated by reductive amination reactions between the Lys and Arg side chains of proteins/peptides and aldehyde groups linked to 7-deazaguanine residues in DNA. These model DPCs were subjected to in vitro replication in the presence of human translesion synthesis DNA polymerases. DPCs containing full-length proteins (11-28 kDa) or a 23-mer peptide blocked human polymerases and . DPC conjugates to a 10-mer peptide were bypassed with nucleotide insertion efficiency 50 -100-fold lower than for native G. Both human polymerase (hPol) and hPol inserted the correct base (C) opposite the 10-mer peptide cross-link, although small amounts of T were added by hPol . Molecular dynamics simulation of an hPol ternary complex containing a template-primer DNA with dCTP opposite the 10-mer peptide DPC revealed that this bulky lesion can be accommodated in the polymerase active site by aligning with the major groove of the adducted DNA within the ternary complex of polymerase and dCTP.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Bypass of DNA-Protein Cross-links Conjugated to the 7-Deazaguanine Position of DNA by Translesion Synthesis Polymerases

Wickramaratne

Mukherjee

et al. 2016

Journal of Biological Chemistry

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“…DNA polymerase I (Pol I) 4 is a high fidelity polymerase belonging to Family A, which includes Taq Pol I, T7 DNA polymerase, and human DNA polymerase ␥, , and (6,25,26). In this work, we identified determinants of Pol I fidelity that do not involve alterations in proofreading by the 3Ј-5Ј exonuclease and that do not cripple the polymerase activity.…”

Section: ϫ5mentioning

confidence: 99%

Highly Tolerated Amino Acid Substitutions Increase the Fidelity of Escherichia coli DNA Polymerase I

Loh¹,

Choe²,

Loeb

2007

Journal of Biological Chemistry

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Fidelity of DNA synthesis, catalyzed by DNA polymerases, is critical for the maintenance of the integrity of the genome. Mutant polymerases with elevated accuracy (antimutators) have been observed, but these mainly involve increased exonuclease proofreading or large decreases in polymerase activity. We have determined the tolerance of DNA polymerase for amino acid substitutions in the active site and in different segments of E. coli DNA polymerase I and have determined the effects of these substitutions on the fidelity of DNA synthesis. We established a DNA polymerase I mutant library, with random substitutions throughout the polymerase domain. This random library was first selected for activity. The essentiality of DNA polymerases and their sequence and structural conservation suggests that few amino acid substitutions would be tolerated. However, we report that two-thirds of single base substitutions were tolerated without loss of activity, and plasticity often occurs at evolutionarily conserved regions. We screened 408 members of the active library for alterations in fidelity of DNA synthesis in Escherichia coli expressing the mutant polymerases and carrying a second plasmid containing a ␤-lactamase reporter. Mutation frequencies varied from 1 ⁄ 1000-to 1000-fold greater compared with wild type. Mutations that produced an antimutator phenotype were distributed throughout the polymerase domain, with 12% clustered in the M-helix. We confirmed that a single mutation in this segment results in increased base discrimination. Thus, this work identifies the M-helix as a determinant of fidelity and suggests that polymerases can tolerate many substitutions that alter fidelity without incurring major changes in activity.DNA polymerases function in DNA replication, repair, and recombination and are essential for maintaining the integrity of the genome. Multiple DNA polymerases have been found in prokaryotes, eukaryotes, and viruses and may have different properties, including variations in the accuracy, or fidelity, of DNA synthesis (1-5). Polymerases that replicate large genomes typically have high fidelity, with error rates on the order of 10 Ϫ5to 10 Ϫ6 (6 -8) to prevent the accumulation of deleterious mutations in the genome. Deficits in DNA polymerase fidelity have been associated with cancer, accelerated aging, and infertility (9 -11).Mutator and antimutator DNA polymerases harbor amino acid substitutions resulting in decreased and increased fidelity, respectively. These variant enzymes provide insights into the structural basis of accurate DNA synthesis (12-18). Many mutators have been identified due to the availability of powerful selection methods (19). The majority of studied mutators harbor amino acid substitutions in well conserved polymerase motifs. Mutator polymerases have also been used extensively in biotechnological applications, such as DNA sequencing and error-prone PCR (20). Far fewer antimutator enzymes have been identified due to the lack of selection methods. The ones identified thus far either exh...

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“…To specifically inactivate the DNA polymerase activity of Pol while leaving the helicase and other potentially important domains intact, we deleted exons 25 (185 base pairs) and 26 (112 base pairs), which encode a conserved aspartic acid residue known to be essential for the polymerase catalytic activity (23,24) and the tyrosine residue that binds the incoming nucleotide (11,14), respectively ( Fig. 1 A and B).…”

Section: Generation Of Mice Specifically Devoid Of Pol Polymerase Actmentioning

confidence: 99%

DNA polymerase θ contributes to the generation of C/G mutations during somatic hypermutation of Ig genes

Masuda

Ouchida

Takeuchi

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

104

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Somatic hypermutation of Ig variable region genes is initiated by activation-induced cytidine deaminase; however, the activity of multiple DNA polymerases is required to ultimately introduce mutations. DNA polymerase (Pol ) has been implicated in mutations at A͞T, but polymerases involved in C͞G mutations have not been identified. We have generated mutant mice expressing DNA polymerase (Pol ) specifically devoid of polymerase activity. Compared with WT mice, Polq-inactive (Polq, the gene encoding Pol ) mice exhibited a reduced level of serum IgM and IgG1. The mutant mice mounted relatively normal primary and secondary immune responses to a T-dependent antigen, but the production of high-affinity specific antibodies was partially impaired. Analysis of the J H4 intronic sequences revealed a slight reduction in the overall mutation frequency in Polq-inactive mice. Remarkably, although mutations at A͞T were unaffected, mutations at C͞G were significantly decreased, indicating an important, albeit not exclusive, role for Pol activity. The reduction of C͞G mutations was particularly focused on the intrinsic somatic hypermutation hotspots and both transitions and transversions were similarly reduced. These findings, together with the recent observation that Pol efficiently catalyzes the bypass of abasic sites, lead us to propose that Pol introduces mutations at C͞G by replicating over abasic sites generated via uracil-DNA glycosylase.abasic site ͉ low-fidelity DNA polymerase ͉ activation-induced cytidine deaminase ͉ uracil-DNA glycosylase

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POLN, a Nuclear PolA Family DNA Polymerase Homologous to the DNA Cross-link Sensitivity Protein Mus308

Cited by 105 publications

References 32 publications

Bypass of DNA-Protein Cross-links Conjugated to the 7-Deazaguanine Position of DNA by Translesion Synthesis Polymerases

Bypass of DNA-Protein Cross-links Conjugated to the 7-Deazaguanine Position of DNA by Translesion Synthesis Polymerases

Highly Tolerated Amino Acid Substitutions Increase the Fidelity of Escherichia coli DNA Polymerase I

DNA polymerase θ contributes to the generation of C/G mutations during somatic hypermutation of Ig genes

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