The Efficiency and Specificity of Apurinic/Apyrimidinic Site Bypass by Human DNA Polymerase η and Sulfolobus solfataricus Dpo4

Kokoska, Robert J.; McCulloch, Scott; Kunkel, Thomas A.

doi:10.1074/jbc.m308515200

Cited by 121 publications

(188 citation statements)

References 40 publications

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“…Noninstructional AP sites are highly mutagenic [93]. However, these could be bypassed by translesion synthesis (TLS) DNA polymerases in the absence of repair [94,95].…”

Section: Multiple Apes Are Also Present In Lower Eukaryotesmentioning

confidence: 99%

Early steps in the DNA base excision/single-strand interruption repair pathway in mammalian cells

2008

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Base excision repair (BER) is an evolutionarily conserved process for maintaining genomic integrity by eliminating several dozen damaged (oxidized or alkylated) or inappropriate bases that are generated endogenously or induced by genotoxicants, predominantly, reactive oxygen species (ROS). BER involves 4-5 steps starting with base excision by a DNA glycosylase, followed by a common pathway usually involving an AP-endonuclease (APE) to generate 3′ OH terminus at the damage site, followed by repair synthesis with a DNA polymerase and nick sealing by a DNA ligase. This pathway is also responsible for repairing DNA single-strand breaks with blocked termini directly generated by ROS. Nearly all glycosylases, far fewer than their substrate lesions particularly for oxidized bases, have broad and overlapping substrate range, and could serve as back-up enzymes in vivo. In contrast, mammalian cells encode only one APE, APE1, unlike two APEs in lower organisms. In spite of overall similarity, BER with distinct subpathways in the mammals is more complex than in E. coli. The glycosylases form complexes with downstream proteins to carry out efficient repair via distinct subpathways one of which, responsible for repair of strand breaks with 3′ phosphate termini generated by the NEIL family glycosylases or by ROS, requires the phosphatase activity of polynucleotide kinase instead of APE1. Different complexes may utilize distinct DNA polymerases and ligases. Mammalian glycosylases have nonconserved extensions at one of the termini, dispensable for enzymatic activity but needed for interaction with other BER and non-BER proteins for complex formation and organelle targeting. The mammalian enzymes are sometimes covalently modified which may affect activity and complex formation. The focus of this review is on the early steps in mammalian BER for oxidized damage.

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“…Noninstructional AP sites are highly mutagenic [93]. However, these could be bypassed by translesion synthesis (TLS) DNA polymerases in the absence of repair [94,95].…”

Section: Multiple Apes Are Also Present In Lower Eukaryotesmentioning

confidence: 99%

Early steps in the DNA base excision/single-strand interruption repair pathway in mammalian cells

2008

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“…However, the absolute number of nucleotides incorporated per DNA binding event varies considerably among Y-family polymerases. For example, recent studies suggest that archaeal Dpo4 is more processive than human Pol (20). Indeed, when replicating circular M13 DNA at high enzyme to template ratios, Dpo4 synthesizes replication products that are several hundred nucleotides in length (Fig.…”

Section: Generation Of Little Finger Domain Chimeras-nativementioning

confidence: 99%

“…4), which have little ability to incorporate a base opposite the 3Ј-T of the dimer. Likewise, Dpo4 can bypass a synthetic abasic site (15,20) (Fig. 4), yet Dbh only does so at high enzyme to template ratios and high levels of dNTPs (17) (Fig.…”

Section: Generation Of Little Finger Domain Chimeras-nativementioning

confidence: 99%

“…At high enzyme to template ratios Dpo4 can synthesize more than 1 kb of DNA, thereby allowing it to substitute for Taq polymerase in PCR assays (15). In addition, the lesion bypass properties of Dpo4 are somewhat like those of the eukaryotic translesion polymerases in that Dpo4 can bypass thymine-thymine cyclobutane pyrimidine dimers (CPDs) (15,18,19) and abasic sites (15,20). In contrast, Dbh is a much more distributive polymerase when replicating undamaged DNA, is unable to incorporate a base opposite a CPD, and bypasses an abasic site with very low efficiency (16,17,21).…”

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confidence: 99%

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Investigating the Role of the Little Finger Domain of Y-family DNA Polymerases in Low Fidelity Synthesis and Translesion Replication

Boudsocq

Kokoska

Plosky

et al. 2004

Journal of Biological Chemistry

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134

164

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Dpo4 and Dbh are Y-family polymerases that originate from two closely related strains of Sulfolobaceae. Quite surprisingly, however, the two polymerases exhibit different enzymatic properties in vitro. For example, Dpo4 can replicate past a variety of DNA lesions, yet Dbh does so with a much lower efficiency. When replicating undamaged DNA, Dpo4 is prone to make base pair substitutions, whereas Dbh predominantly makes single-base deletions. Overall, the two proteins are 54% identical, but the greatest divergence is found in their respective little finger (LF) domains, which are only 41% identical. To investigate the role of the LF domain in the fidelity and lesion-bypassing abilities of Y-family polymerases, we have generated chimeras of Dpo4 and Dbh in which their LF domains have been interchanged. Interestingly, by replacing the LF domain of Dbh with that of Dpo4, the enzymatic properties of the chimeric enzyme are more Dpo4-like in that the enzyme is more processive, can bypass an abasic site and a thymine-thymine cyclobutane pyrimidine dimer, and predominantly makes base pair substitutions when replicating undamaged DNA. The converse is true for the Dpo4-LF-Dbh chimera, which is more Dbh-like in its processivity and ability to bypass DNA adducts and generate single-base deletion errors. Our studies indicate that the unique but variable LF domain of Y-family polymerases plays a major role in determining the enzymatic and biological properties of each individual Y-family member.

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“…To gain a better understanding of the mechanistic aspects of the second-gate mutation E664K on RNA synthesis by TGK, we measured steady-state NTP incorporation kinetics, single-hit extension, and termination probabilities as a measure of polymerase processivity (27) and primer/ template affinities for the parent polymerase TgoT, for TGK, and for the intermediate variants TGE and TYK.…”

Section: Synthesis Of Protein Coding and Functionalized Mrnasmentioning

confidence: 99%

A short adaptive path from DNA to RNA polymerases

Cozens

Pinheiro

Vaisman

et al. 2012

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

115

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DNA polymerase substrate specificity is fundamental to genome integrity and to polymerase applications in biotechnology. In the current paradigm, active site geometry is the main site of specificity control. Here, we describe the discovery of a distinct specificity checkpoint located over 25 Å from the active site in the polymerase thumb subdomain. In Tgo, the replicative DNA polymerase from Thermococcus gorgonarius, we identify a single mutation (E664K) within this region that enables translesion synthesis across a template abasic site or a cyclobutane thymidine dimer. In conjunction with a classic "steric-gate" mutation (Y409G) in the active site, E664K transforms Tgo DNA polymerase into an RNA polymerase capable of synthesizing RNAs up to 1.7 kb long as well as fully pseudouridine-, 5-methyl-C-, 2′-fluoro-, or 2′-azido-modified RNAs primed from a wide range of primer chemistries comprising DNA, RNA, locked nucleic acid (LNA), or 2′O-methyl-DNA. We find that E664K enables RNA synthesis by selectively increasing polymerase affinity for the noncognate RNA/DNA duplex as well as lowering the K m for ribonucleotide triphosphate incorporation. This gatekeeper mutation therefore identifies a key missing step in the adaptive path from DNA to RNA polymerases and defines a previously unknown postsynthetic determinant of polymerase substrate specificity with implications for the synthesis and replication of noncognate nucleic acid polymers.processivity | protein engineering | second gate R eplicative polymerases require extraordinary specificity in substrate selection, incorporation, and replication both to ensure fidelity and to exclude noncognate and/or damaged nucleotides from the genome. A particular threat to DNA genome integrity are ribonucleotide triphosphates (NTPs), which are present in the cell at concentrations up to 100-fold in excess of the cognate deoxyribonucleotide triphosphates (dNTPs) (1-3) yet differ from them only by the presence of a 2′-hydroxyl(-OH) group. Indeed, although DNA polymerases have evolved to exclude NTPs from their active sites, incorporation does occur to a detectable degree, with significant implications for genome stability and repair (2, 4). This issue may be even more acute for thermophilic organisms, because high temperatures further increase genome instability by accelerating the spontaneous degradation of RNA (5). Control of NTP incorporation by DNA polymerases is therefore a paradigmatic case of the link between polymerase substrate specificity and genome stability.DNA polymerases from all three domains of life are known to use a common strategy to prevent NTP incorporation into the nascent strand, by exerting stringent geometric control of the chemical nature of the 2′ position of the incoming nucleotide through a single active site residue, the "steric gate" (6). This strategy is so efficient that mutation of the steric gate alone (e.g., to an amino acid with a smaller side chain) can reduce discrimination against NTP incorporation by several orders of magnitude (6-13). Howev...

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The Efficiency and Specificity of Apurinic/Apyrimidinic Site Bypass by Human DNA Polymerase η and Sulfolobus solfataricus Dpo4

Cited by 121 publications

References 40 publications

Early steps in the DNA base excision/single-strand interruption repair pathway in mammalian cells

Early steps in the DNA base excision/single-strand interruption repair pathway in mammalian cells

Investigating the Role of the Little Finger Domain of Y-family DNA Polymerases in Low Fidelity Synthesis and Translesion Replication

A short adaptive path from DNA to RNA polymerases

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