DNA Polymerase Locks Replication Fork Under Stress

Abstract

Replication of DNA requires the parental DNA to be unwound to allow the genetic information to be faithfully duplicated by the replisome. While this function is usually shared by a host of proteins in the replisome, notably DNA polymerase (DNAP) and helicase, the consequence of DNAP synthesizing DNA while decoupled from helicase remains not well understood. The unwinding of downstream DNA poses significant stress to DNAP, and the interaction between DNAP and the replication fork may affect replication restart. In this work, we examined the consequences of DNAP working against the stress of the DNA replication fork. We found that prolonged exposure of DNAP to the stress of the replication fork inactivates replication. Surprisingly, replication inactivation was often accompanied by a strong DNAP interaction with the leading and lagging strands at the fork, locking the fork in place. We demonstrated that fork locking is a consequence of DNAP forward translocation, and the exonuclease activity of DNAP, which allows DNAP to move in reverse, is essential in protecting the fork from inactivation. Furthermore, we found the locking configuration is not reversible by the subsequent addition of helicase. Collectively, this study provides a deeper understanding of the DNAP-fork interaction and mechanism in keeping the replication fork active during replication stress.

Figure 1.

DNAP can lock the replication fork. a. Experimental techniques for investigating DNAP interactions with replication fork. First, the unzipping tracker uses the DNA fork to track the trajectory of the DNAP in real-time. Active forks held under a constant force result in an increase in the tether extension while inactive forks do not increase the extension. Subsequently, the unzipping mapper detects the location and strength of a bound protein interacting with the fork via mechanical unzipping through the remaining DNA. A locked fork results in a force rise at the start of the unzipping mapper step while an unlocked fork has no detectable force rise. b. Representative active (blue) and inactive (red) traces during the unzipping tracker step. The replication distance of the DNAP is tracked for 90 s before proceeding to the unzipping mapper step. c. Immediately after the unzipping tracker step, the unzipping mapper is employed on the same traces shown in b to detect any interactions of DNAP with the remaining parental DNA. The unzipping fork position at the start of the unzipping mapper corresponds to the fork position after replication in the unzipping tracker step. At this initial unzipping fork position, the initial unzipping force indicates resistance to strand opening for the inactive trace (red) and minimal resistance for the active trace (blue). The unzipping mapper curve of naked DNA in the absence of the DNAP is shown in black. d. The initial unzipping force from the beginning of the unzipping mapper versus the replication distance during the unzipping tracker from N = 137 individual traces. Traces identified as active during the replication tracker are shown in blue, and traces identified as inactive in red. e. The inactive fraction versus incubation duration of DNAP with DNA forks. Error bars represent the standard deviation, with N = 41, 44, 38, and 41 individual traces for 26, 90, 147, and 210 min incubation, respectively.

Figure 2.

DNAP exonuclease activity limits fork-locking. The unzipping tracker and unzipping mapper techniques are used to investigate the role of the exonuclease activity of DNAP on fork-locking. Two exonuclease-deficient DNAP mutants, exo- DNAP and Sequence, are investigated after a 90 ± 17 min incubation with the fork. a. Representative traces of exo- DNAP (green) and Sequenase (magenta) during the unzipping tracker step. b. Histograms of the replication distance at the beginning of the unzipping tracker step. The histograms have N = 43, 90, and 43 individual traces for WT DNAP, exo- DNAP, and Sequenase, respectively, with the mean and standard deviation indicated. c. The inactive fraction and the initial unzipping force of the three DNAPs. Top panel: Error bars represent the standard deviation. The numbers of traces for inactive fractions are the same as in b. Bottom panel: The initial unzipping forces have N = 34, 87, and 43 individual traces for WT DNAP, exo- DNAP, and Sequenase, respectively. Active forks are colored blue, and inactive forks are colored red.

Figure 3.

DNAP forward translocation promotes fork-locking The unzipping tracker and unzipping mapper techniques are used to investigate the role of the forward translocation of DNAP on fork-locking. For these experiments, exo- DNAP was used as it has significant fork-locking activity. a. Representative traces of exo- DNAP with reduced dNTP concentrations during the unzipping tracker step. b. Replication rate of the exo- DNAP versus dNTP concentration. Data are from N = 41, 35, 30, and 20 individual traces with 5, 25, 75, and 1000 µM of each dNTP, respectively. Error bars represent the standard errors of the means. c. The inactive fraction of the exo- DNAP at different dNTP concentrations, after a 90 ± 17 min incubation with the fork. Error bars represent the standard deviation. Data are from N = 20, 23, 21, and 90 individual traces for 5, 25, 75 and 1000 µM of each dNTP, respectively. d. The initial unzipping force of the exo- DNAP at different dNTP concentrations, after a 90 ± 17 min incubation with the fork. Traces that were active during the unzipping tracker are shown in blue, while inactive traces are shown in red, while traces in the absence of dNTPs and with ddNTPS shown in black did not have a tracking step. Data are from N = 63, 20, 20, 21, and 87 individual traces for 0, 5, 25, 75, and 1000 µM of each dNTP, respectively. The ddNTP 1000 µM condition contains N = 62 individual traces.

Figure 4.

Locked replication forks cannot be readily unlocked. a. The inactive fraction and initial unzipping force of WT and exo- DNAP under different protein concentrations. Forks were initially incubated with the indicated DNAP type and concentration for 90 min, and subsequently, the DNAP type and concentration were changed to the indicated subsequent condition. Error bars represent the standard deviations. Inactive fraction traces: N = 44, 33, 33, 90, 42, and 48 for conditions shown from left to right. Initial unzipping force traces: N = 34, 28, 27, 87, 40, and 47 for conditions shown from left to right b. The fraction remaining locked after helicase is introduced to the forks. After the initial incubation of 90 min with WT or exo- DNAP, tethers with locked forks were selected (Methods). After the addition of helicase, these locked forks were checked using unzipping tracker and mapper assays. Reactivated forks are shown in blue, and forks remaining inactive are shown in red. Error bars represent the standard deviations. Fraction remaining locked traces: N = 24 and 24 for conditions shown from left to right. Initial unzipping force traces: N = 22 and 23 for conditions shown from left to right.