Abstract
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.
Highlights
Chromosome duplication is performed by a multiprotein replisome machine that simultaneously replicates both strands of the parental duplex [1,2,3,4,5]
Primase has a negative effect on processivity in the presence of rNTPs provided that lagging strand synthesis is not permitted, but primase does not appear to affect processivity in the absence of rNTPs whether the lagging strand is synthesized or not
The primase effect is masked during coupled synthesis, because the extra clamps on DNA required for lagging strand replication provide ample extra grip to overcome replisome instability caused by primase
Summary
Chromosome duplication is performed by a multiprotein replisome machine that simultaneously replicates both strands of the parental duplex [1,2,3,4,5]. DNA polymerases only extend DNA in the 3 –5 direction, and the antiparallel geometry of duplex DNA requires the two strands to be synthesized in opposite directions [3]. The leading strand polymerase extends DNA in the same direction as helicase unwinding, while the lagging strand is copied in the opposite direction and is synthesized as multiple Okazaki fragments. Formation of DNA loops during replication has gained much experimental support in studies of replisomes from various sources [12,13,14,15] Any of these lagging strand specific events: priming, SSB displacement, and formation of replication loops, could conceivably take a toll on the processivity and/or rate of the replication fork
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