Replisome mechanics: lagging strand events that influence speed and processivity.
The antiparallel structure of DNA requires lagging strand synthesis to proceed in the opposite direction of the replication fork. This imposes unique events that occur only on the lagging strand, such as primase binding to DnaB helicase, RNA synthesis, and SS B antigen (SSB) displacement during Okazaki fragment extension. Single-molecule and ensemble techniques are combined to examine the effect of lagging strand events on the Escherichia coli replisome rate and processivity. We find that primase activity lowers replisome processivity but only when lagging strand extension is inoperative. rNTPs also lower replisome processivity. However, the negative effects of primase and rNTPs on processivity are overcome by the extra grip on DNA provided by the lagging strand polymerases. Visualization of single molecules reveals that SSB accumulates at forks and may wrap extensive amounts of single-strand DNA. Interestingly SSB has an inter-strand positive effect on the rate of the leading strand based in its interaction with the replicase χ-subunit. Further, the lagging strand polymerase is faster than leading strand synthesis, indicating that replisome rate is limited by the helicase. Overall, lagging strand events that impart negative effects on the replisome are counterbalanced by the positive effects of SSB and additional sliding clamps during Okazaki fragment extension.
Howard Hughes Medical Institute, Rockefeller University, 1230 York Avenue, NY 10065, USA.
Roxana E
RE
Georgescu
Howard Hughes Medical Institute, Rockefeller University, 1230 York Avenue, NY 10065, USA.
Nina
N
Yao
Manhattan College, 4513 Manhattan College Pkwy, Riverdale, NY 10471, USA.
Chiara
C
Indiani
Howard Hughes Medical Institute, Rockefeller University, 1230 York Avenue, NY 10065, USA.
Olga
O
Yurieva
Howard Hughes Medical Institute, Rockefeller University, 1230 York Avenue, NY 10065, USA odonnel@rockefeller.edu.
Mike E
ME
O'Donnell
D001324:Autoantigens / Q000378:metabolism; D004247:DNA / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D019915:DNA Primase / Q000378:metabolism; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D009097:Multienzyme Complexes / Q000378:metabolism*; D012261:Ribonucleoproteins / Q000378:metabolism; D012265:Ribonucleotides / Q000378:metabolism; D013045:Species Specificity; D000097585:SS-B Antigen; D000099272:Replisomes
D001324:Autoantigens; D004247:DNA; D019915:DNA Primase; D004259:DNA-Directed DNA Polymerase; D009097:Multienzyme Complexes; D012261:Ribonucleoproteins; D012265:Ribonucleotides; D000097585:SS-B Antigen; D000099272:Replisomes; C022262:Okazaki fragments