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Dynamics of enzymatic interactions during short flap human Okazaki fragment processing by two forms of human DNA polymerase ?.


ABSTRACT: Lagging strand DNA replication requires the concerted actions of DNA polymerase ?, Fen1 and DNA ligase I for the removal of the RNA/DNA primers before ligation of Okazaki fragments. To better understand this process in human cells, we have reconstituted Okazaki fragment processing by the short flap pathway in vitro with purified human proteins and oligonucleotide substrates. We systematically characterized the key events in Okazaki fragment processing: the strand displacement, Pol ?/Fen1 combined reactions for removal of the RNA/DNA primer, and the complete reaction with DNA ligase I. Two forms of human DNA polymerase ? were studied: Pol ?4 and Pol ?3, which represent the heterotetramer and the heterotrimer lacking the p12 subunit, respectively. Pol ?3 exhibits very limited strand displacement activity in contrast to Pol ?4, and stalls on encounter with a 5'-blocking oligonucleotide. Pol ?4 and Pol ?3 exhibit different characteristics in the Pol ?/Fen1 reactions. While Pol ?3 produces predominantly 1 and 2 nt cleavage products irrespective of Fen1 concentrations, Pol ?4 produces cleavage fragments of 1-10 nts at low Fen1 concentrations. Pol ?3 and Pol ?4 exhibit comparable formation of ligated products in the complete system. While both are capable of Okazaki fragment processing in vitro, Pol ?3 exhibits ideal characteristics for a role in Okazaki fragment processing. Pol ?3 readily idles and in combination with Fen1 produces primarily 1 nt cleavage products, so that nick translation predominates in the removal of the blocking strand, avoiding the production of longer flaps that require additional processing. These studies represent the first analysis of the two forms of human Pol ? in Okazaki fragment processing. The findings provide evidence for the novel concept that Pol ?3 has a role in lagging strand synthesis, and that both forms of Pol ? may participate in DNA replication in higher eukaryotic cells.

SUBMITTER: Lin SH 

PROVIDER: S-EPMC3825817 | biostudies-literature | 2013 Nov

REPOSITORIES: biostudies-literature

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Dynamics of enzymatic interactions during short flap human Okazaki fragment processing by two forms of human DNA polymerase δ.

Lin Szu Hua Sharon SH   Wang Xiaoxiao X   Zhang Sufang S   Zhang Zhongtao Z   Lee Ernest Y C EY   Lee Marietta Y W T MY  

DNA repair 20130910 11


Lagging strand DNA replication requires the concerted actions of DNA polymerase δ, Fen1 and DNA ligase I for the removal of the RNA/DNA primers before ligation of Okazaki fragments. To better understand this process in human cells, we have reconstituted Okazaki fragment processing by the short flap pathway in vitro with purified human proteins and oligonucleotide substrates. We systematically characterized the key events in Okazaki fragment processing: the strand displacement, Pol δ/Fen1 combine  ...[more]

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