title: Roles of DNA polymerase I in leading and lagging-strand replication defined by a high-resolution mutation footprint of ColE1 plasmid replication.
abstract: DNA polymerase I (pol I) processes RNA primers during lagging-strand synthesis and fills small gaps during DNA repair reactions. However, it is unclear how pol I and pol III work together during replication and repair or how extensive pol I processing of Okazaki fragments is in vivo. Here, we address these questions by analyzing pol I mutations generated through error-prone replication of ColE1 plasmids. The data were obtained by direct sequencing, allowing an accurate determination of the mutation spectrum and distribution. Pol I's mutational footprint suggests: (i) during leading-strand replication pol I is gradually replaced by pol III over at least 1.3 kb; (ii) pol I processing of Okazaki fragments is limited to ∼20 nt and (iii) the size of Okazaki fragments is short (∼250 nt). While based on ColE1 plasmid replication, our findings are likely relevant to other pol I replicative processes such as chromosomal replication and DNA repair, which differ from ColE1 replication mostly at the recruitment steps. This mutation footprinting approach should help establish the role of other prokaryotic or eukaryotic polymerases in vivo, and provides a tool to investigate how sequence topology, DNA damage, or interactions with protein partners may affect the function of individual DNA polymerases.
authors.affiliation: Department of Microbiology and Environmental Toxicology, University of California Santa Cruz, 1156 High Street Santa Cruz, CA 95060, USA.
authors.forename: Jennifer M
authors.initials: JM
authors.lastname: Allen
authors.forename: David M
authors.initials: DM
authors.lastname: Simcha
authors.forename: Nolan G
authors.initials: NG
authors.lastname: Ericson
authors.forename: David L
authors.initials: DL
authors.lastname: Alexander
authors.forename: Jacob T
authors.initials: JT
authors.lastname: Marquette
authors.forename: Benjamin P
authors.initials: BP
authors.lastname: Van Biber
authors.forename: Chris J
authors.initials: CJ
authors.lastname: Troll
authors.forename: Rachel
authors.initials: R
authors.lastname: Karchin
authors.forename: Jason H
authors.initials: JH
authors.lastname: Bielas
authors.forename: Lawrence A
authors.initials: LA
authors.lastname: Loeb
authors.forename: Manel
authors.initials: M
authors.lastname: Camps
mesh_terms: D001483:Base Sequence; D004247:DNA / Q000378:metabolism; D018983:DNA Footprinting; D004256:DNA Polymerase I / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D004261:DNA Replication; D030561:Databases, Nucleic Acid; D009154:Mutation; D010957:Plasmids / Q000096:biosynthesis* / Q000737:chemistry
chemical_list: C022262:Okazaki fragments; D004247:DNA; D004256:DNA Polymerase I