The Roles of Family B and D DNA Polymerases in Thermococcus species 9°N Okazaki Fragment Maturation.

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TitleThe Roles of Family B and D DNA Polymerases in Thermococcus species 9°N Okazaki Fragment Maturation.
Publication TypeJournal Article
Year of Publication2015
AuthorsGreenough, L, Kelman, Z, Gardner, AF
JournalJ Biol Chem
Date Published2015 Mar 26
ISSN1083-351X
Abstract<p>During replication, Okazaki fragment maturation is a fundamental process that joins discontinuously synthesized DNA fragments into a contiguous lagging strand. Efficient maturation prevents repeat sequence expansions, small duplications and generation of double-stranded DNA breaks. To address the components required for the process in Thermococcus, Okazaki fragment maturation was reconstituted in vitro using purified proteins from Thermococcus species 9°N or cell extracts. A dual color fluorescence assay was developed to monitor reaction substrates, intermediates and products. DNA polymerase D (polD) was proposed to function as the replicative polymerase in Thermococcus replicating both the leading and the lagging strand. It is shown here however that it stops before the previous Okazaki fragments, failing to rapidly process them. Instead, Family B DNA polymerase (polB) was observed to rapidly fill the gaps left by polD and displaces the downstream Okazaki fragment to create a flap structure. This flap structure was cleaved by flap endonuclease 1 (Fen1) and the resultant nick was ligated by DNA ligase to form a mature lagging strand. The similarities to both bacterial and eukaryal systems and evolutionary implications of archaeal Okazaki fragment maturation are discussed.</p>
DOI10.1074/jbc.M115.638130
Alternate JournalJ. Biol. Chem.
PubMed ID25814667