DNA Ligase I and Proliferating Cell Nuclear Antigen Form a Functional Complex

Tom, Samson; Henricksen, Leigh A.; Park, Min; Bambara, Robert A.

doi:10.1074/jbc.m101673200

Cited by 100 publications

(100 citation statements)

References 64 publications

(100 reference statements)

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“…IS, internal standard insertion of C opposite 8oxoG, is performed by Pol d/e. Repair of this strand is then completed by the PCNA stimulated sealing by DNA ligase I (Tom et al, 2001). The formation of 8oxoG/C pairs during processing of 8oxoG/A mispairs by Pol d/e-dependent BER changes the repair mode for this oxidized guanine.…”

Section: Discussionmentioning

confidence: 99%

Base excision repair of adenine/8-oxoguanine mispairs by an aphidicolin-sensitive DNA polymerase in human cell extracts

et al. 2002

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Replication of DNA containing 8-oxo-7,8-dihydroguanine (8oxoG) can generate 8oxoG/A base pairs which, if uncorrected, lead to G?T transversions. It is generally accepted that the repair of these promutagenic base pairs in human cells is initiated by the MutY DNA glycosylase homolog (hMYH). Here we provide biochemical evidence that human cell extracts perform base excision repair (BER) on both DNA strands of an 8oxoG/A mismatch. At early repair times the specificity of nucleotide incorporation indicates a preferential insertion of C opposite 8oxoG leading to the formation of 8oxoG/C pairs. This is followed by repair synthesis on the opposite DNA strand that is consistent with hOGG1-mediated correction of 8oxoG/C to G/C. Repair synthesis on either strand is completely inhibited by aphidicolin suggesting that a replicative DNA polymerase is involved in the gap filling. This is the first demonstration that repair of 8oxoG/A base pairs is by two BER events likely mediated by Pold/e. We suggest that the Pold/emediated BER is the general mode of repair when BER lesions are formed at replication forks.

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Section: Discussionmentioning

confidence: 99%

Base excision repair of adenine/8-oxoguanine mispairs by an aphidicolin-sensitive DNA polymerase in human cell extracts

et al. 2002

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“…The current model proposed for Okazaki fragment processing indicates that it is a sequential pathway (33)(34)(35). Proliferating cell nuclear antigen (PCNA) appears to act as a coordination factor at the replication fork and interacts with different enzymes in a stepwise manner.…”

mentioning

confidence: 99%

Cleavage Specificity of Saccharomyces cerevisiae Flap Endonuclease 1 Suggests a Double-Flap Structure as the Cellular Substrate

Kao

Henricksen²,

Liu

et al. 2002

Journal of Biological Chemistry

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139

161

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Flap endonuclease 1 (FEN1) is a structure-specific nuclease that cleaves substrates containing unannealed 5-flaps during Okazaki fragment processing. Cleavage removes the flap at or near the point of annealing. The preferred substrate for archaeal FEN1 or the 5-nuclease domains of bacterial DNA polymerases is a double-flap structure containing a 3-tail on the upstream primer adjacent to the 5-flap. We report that FEN1 in Saccharomyces cerevisiae (Rad27p) exhibits a similar specificity. Cleavage was most efficient when the upstream primer contained a 1-nucleotide 3-tail as compared with the fully annealed upstream primer traditionally tested. The site of cleavage was exclusively at a position one nucleotide into the annealed region, allowing human DNA ligase I to seal all resulting nicks. In contrast, a portion of the products from traditional flap substrates is not ligated. The 3-OH of the upstream primer is not critical for double-flap recognition, because Rad27p is tolerant of modifications. However, the positioning of the 3-nucleotide defines the site of cleavage. We have tested substrates having complementary tails that equilibrate to many structures by branch migration. FEN1 only cleaved those containing a 1-nucleotide 3-tail. Equilibrating substrates containing 12-ribonucleotides at the end of the 5-flap simulates the situation in vivo. Rad27p cleaves this substrate in the expected 1-nucleotide 3-tail configuration. Overall, these results suggest that the double-flap substrate is formed and cleaved during eukaryotic DNA replication in vivo.Pathways for DNA replication, recombination, and repair are all proposed to involve DNA flap intermediates (1-8). The creation and resolution of single-stranded flap structures is a preferred method for removal of damaged or mismatched nucleotides. DNA replication requires the synthesis and joining of discontinuous segments, or Okazaki fragments. Each fragment is initiated by an RNA primer that must be removed prior to joining. Synthesis from the upstream fragment is thought to displace the RNA primer and some adjacent DNA into a single-stranded flap (8 -11). Processing of the flap intermediate is carried out by the structure-specific flap endonuclease 1 or FEN1 1 (4).FEN1 is evolutionarily conserved, and it has intrinsic 5Ј-3Ј-exonuclease and endonuclease activities (4,5,(12)(13)(14). The exonuclease activity utilizes double-stranded DNA with a nick or gap, whereas the endonuclease activity requires a flap structure. In prokarya, the FEN1 homologue is the 5Ј-nuclease domain associated with DNA polymerase I; however, FEN1 exists as a separate polypeptide in eukarya, archaea, and some bacteriophages (15). The mechanism and substrate specificity of FEN1 have been studied by many groups (4, 15-23). The preferred substrate for endonucleolytic cleavage was defined as a nick-flap substrate in vitro. It consists of upstream and downstream primers annealed to a template in a manner that creates a nick. The downstream primer contains a single-stranded 5Ј-flap region (4, 24).Sever...

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“…The ring‐shaped structure of PCNA and the IDCL is conserved among PCNAs from H. sapiens , Saccharomyces cerevisiae , and E. histolytica 3, 4. As the role of PCNA in the catalytic enhancement of the nick‐sealing step by DNA ligases is controversial 16, 21, we tested if an orthologous PCNA from H. sapiens would influence the nick‐sealing activity of EhDNAligI. HsPCNA does not influence the nick‐sealing rate of full‐length (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…PCNA stimulates the enzymatic activities of various enzymes that contain the PIP box interacting motif like Ape2, XPF, and FEN1 21, 38, 39. All these proteins share canonical PIP box sequences that promote their interaction with PCNA.…”

Section: Discussionmentioning

confidence: 99%

Proliferating cell nuclear antigen restores the enzymatic activity of a DNA ligase I deficient in DNA binding

Trasviña-Arenas¹,

Cardona-Félix²,

Azuara-Licéaga

et al. 2017

FEBS Open Bio

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Proliferating cell nuclear antigen (PCNA) coordinates multienzymatic reactions by interacting with a variety of protein partners. Family I DNA ligases are multidomain proteins involved in sealing of DNA nicks during Okazaki fragment maturation and DNA repair. The interaction of DNA ligases with the interdomain connector loop (IDCL) of PCNA through its PCNA‐interacting peptide (PIP box) is well studied but the role of the interacting surface between both proteins is not well characterized. In this work, we used a minimal DNA ligase I and two N‐terminal deletions to establish that DNA binding and nick‐sealing stimulation of DNA ligase I by PCNA are not solely dependent on the PIP box–IDCL interaction. We found that a truncated DNA ligase I with a deleted PIP box is stimulated by PCNA. Furthermore, the activity of a DNA ligase defective in DNA binding is rescued upon PCNA addition. As the rate constants for single‐turnover ligation for the full‐length and truncated DNA ligases are not affected by PCNA, our data suggest that PCNA stimulation is achieved by increasing the affinity for nicked DNA substrate and not by increasing catalytic efficiency. Surprisingly C‐terminal mutants of PCNA are not able to stimulate nick‐sealing activity of Entamoeba histolytica DNA ligase I. Our data support the notion that the C‐terminal region of PCNA may be involved in promoting an allosteric transition in E. histolytica DNA ligase I from a spread‐shaped to a ring‐shaped structure. This study suggests that the ring‐shaped PCNA is a binding platform able to stabilize coevolved protein–protein interactions, in this case an interaction with DNA ligase I.

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DNA Ligase I and Proliferating Cell Nuclear Antigen Form a Functional Complex

Cited by 100 publications

References 64 publications

Base excision repair of adenine/8-oxoguanine mispairs by an aphidicolin-sensitive DNA polymerase in human cell extracts

Base excision repair of adenine/8-oxoguanine mispairs by an aphidicolin-sensitive DNA polymerase in human cell extracts

Cleavage Specificity of Saccharomyces cerevisiae Flap Endonuclease 1 Suggests a Double-Flap Structure as the Cellular Substrate

Proliferating cell nuclear antigen restores the enzymatic activity of a DNA ligase I deficient in DNA binding

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