The Werner Syndrome Helicase/Exonuclease Processes Mobile D-Loops through Branch Migration and Degradation

Abstract

RecQ DNA helicases are critical for preserving genome integrity. Of the five RecQ family members identified in humans, only the Werner syndrome protein (WRN) possesses exonuclease activity. Loss of WRN causes the progeroid disorder Werner syndrome which is marked by cancer predisposition. Cellular evidence indicates that WRN disrupts potentially deleterious intermediates in homologous recombination (HR) that arise in genomic and telomeric regions during DNA replication and repair. Precisely how the WRN biochemical activities process these structures is unknown, especially since the DNA unwinding activity is poorly processive. We generated biologically relevant mobile D-loops which mimic the initial DNA strand invasion step in HR to investigate whether WRN biochemical activities can disrupt this joint molecule. We show that WRN helicase alone can promote branch migration through an 84 base pair duplex region to completely displace the invading strand from the D-loop. However, substrate processing is altered in the presence of the WRN exonuclease activity which degrades the invading strand both prior to and after release from the D-loop. Furthermore, telomeric D-loops are more refractory to disruption by WRN, which has implications for tighter regulation of D-loop processing at telomeres. Finally, we show that WRN can recognize and initiate branch migration from both the 5′ and 3′ ends of the invading strand in the D-loops. These findings led us to propose a novel model for WRN D-loop disruption. Our biochemical results offer an explanation for the cellular studies that indicate both WRN activities function in processing HR intermediates.

Understanding the molecular basis of common fragile sites instability: Role of the proteins involved in the recovery of stalled replication forks

“…56,58,59 Although how each biochemical activity of WRN contributes to its function is not fully clarified, several studies indicate specific involvement in different metabolic processes. [60][61][62][63][64][65][66][67] Moreover, WS cells have an elevated spontaneous yield of DNA breakage, which is consistent with the observed high rate of chromosomal rearrangements, 56,68 and depends on an alternative pathway, probably involving break-induced replication, to recover stalled replication forks. 69 These findings support the hypothesis that, upon replication fork stalling, WRN acts to limit DSBs and fork collapse or to promote error-free repair of DSBs.…”

Replication Protein A Stimulates the Werner Syndrome Protein Branch Migration Activity

“…Proteins-Recombinant hexahistidine-tagged WRN protein and the exonuclease-dead E84A mutant (X-WRN) were purified as previously described (38,46). The concentration of active protein was determined by a Bradford assay (Bio-Rad) and standard helicase activity assay with a 16-bp forked duplex.…”

“…The plasmids used for making the 84-bp non-telomeric and telomeric D-loops were as previously described (38) and were purified by two rounds of ethidium bromide-saturated CsCl equilibrium gradient ultracentrifugation (Lofstrand Labs, Gaithersburg, MD). The plasmid-based D-loop sub-strates (Fig.…”

“…The plasmid-based D-loop sub-strates (Fig. 1B) were constructed as described previously (38). Briefly, RecA (4 M) was incubated with the invading strand oligonucleotide (3.6 M nucleotide) for 5 min at 37°C, and then the supercoiled plasmid (300 M nucleotides) was added and incubated for an additional 3 min.…”

“…However, WRN possesses other catalytic activities that may be more relevant to processing HR intermediates and replication fork remodeling. WRN exhibits 3Ј to 5Ј exonuclease activity (35) and possesses single strand DNA annealing (36) and branch migration (BM) activities (37,38). WRN, BLM, and RECQ1 all promote BM of mobile plasmid-based D-loops (Fig.…”