title: The roles of family B and D DNA polymerases in Thermococcus species 9°N Okazaki fragment maturation.
abstract: 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 strands. 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 eukaryotic systems and evolutionary implications of archaeal Okazaki fragment maturation are discussed.
authors: From New England Biolabs, Inc., Ipswich, Massachusetts 01938 and.
authors: Lucia
authors: L
authors: Greenough
authors: the National Institute of Standards and Technology, Rockville, Maryland 20850.
authors: Zvi
authors: Z
authors: Kelman
authors: From New England Biolabs, Inc., Ipswich, Massachusetts 01938 and gardner@neb.com.
authors: Andrew F
authors: AF
authors: Gardner
mesh_terms: D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004258:DNA Polymerase III / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019951:DNA Polymerase beta / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004261:DNA Replication / Q000502:physiology; D019641:DNA, Archaeal / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D045585:Flap Endonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019712:Thermococcus / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D019843:Archaeal Proteins; D019641:DNA, Archaeal; C022262:Okazaki fragments; D004247:DNA; D004258:DNA Polymerase III; D019951:DNA Polymerase beta; D045585:Flap Endonucleases
keywords: DNA; DNA enzyme; DNA polymerase; DNA replication; DNA synthesis; Okazaki fragment; lagging strand; nucleic acid enzymology; replisome