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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]

PMID: 24035200

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Project description:RNA-DNA hybrids are common in chromosomal DNA. Persistent RNA-DNA hybrids result in replication fork stress, DNA breaks, and neurological disorders in humans. During replication, Okazaki fragment synthesis relies on frequent RNA primer placement, providing one of the most prominent forms of covalent RNA-DNA strands in vivo The mechanism of Okazaki fragment maturation, which involves RNA removal and subsequent DNA replacement, in bacteria lacking RNase HI remains unclear. In this work, we reconstituted repair of a linear model Okazaki fragment in vitro using purified recombinant enzymes from Bacillus subtilis We showed that RNase HII and HIII are capable of incision on Okazaki fragments in vitro and that both enzymes show mild stimulation by single-stranded DNA binding protein (SSB). We also showed that RNase HIII and DNA polymerase I provide the primary pathway for Okazaki fragment maturation in vitro Furthermore, we found that YpcP is a 5' to 3' nuclease that can act on a wide variety of RNA- and DNA-containing substrates and exhibits preference for degrading RNA in model Okazaki fragments. Together, our data showed that RNase HIII and DNA polymerase I provide the primary pathway for Okazaki fragment maturation, whereas YpcP also contributes to the removal of RNA from an Okazaki fragment in vitroIMPORTANCE All cells are required to resolve the different types of RNA-DNA hybrids that form in vivo When RNA-DNA hybrids persist, cells experience an increase in mutation rate and problems with DNA replication. Okazaki fragment synthesis on the lagging strand requires an RNA primer to begin synthesis of each fragment. The mechanism of RNA removal from Okazaki fragments remains unknown in bacteria that lack RNase HI. We examined Okazaki fragment processing in vitro and found that RNase HIII in conjunction with DNA polymerase I represent the most efficient repair pathway. We also assessed the contribution of YpcP and found that YpcP is a 5' to 3' exonuclease that prefers RNA substrates with activity on Okazaki and flap substrates in vitro.

Dataset Information

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

Publications

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

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