Single-molecule visualization of human BLM helicase as it acts upon double- and single-stranded DNA substrates

Abstract

Bloom helicase (BLM) and its orthologs are essential for the maintenance of genome integrity. BLM defects represent the underlying cause of Bloom Syndrome, a rare genetic disorder that is marked by strong cancer predisposition. BLM deficient cells accumulate extensive chromosomal aberrations stemming from dysfunctions in homologous recombination (HR). BLM participates in several HR stages and helps dismantle potentially harmful HR intermediates. However, much remains to be learned about the molecular mechanisms of these BLM-mediated regulatory effects. Here, we use DNA curtains to directly visualize the activity of BLM helicase on single molecules of DNA. Our data show that BLM is a robust helicase capable of rapidly (∼70–80 base pairs per second) unwinding extensive tracts (∼8–10 kilobases) of double-stranded DNA (dsDNA). Importantly, we find no evidence for BLM activity on single-stranded DNA (ssDNA) that is bound by replication protein A (RPA). Likewise, our results show that BLM can neither associate with nor translocate on ssDNA that is bound by the recombinase protein RAD51. Moreover, our data reveal that the presence of RAD51 also blocks BLM translocation on dsDNA substrates. We discuss our findings within the context of potential regulator roles for BLM helicase during DNA replication and repair.

Single-molecule visualization of human RECQ5 interactions with single-stranded DNA recombination intermediates

“…Interestingly, although human BLM is considered an antirecombinase ( 3 , 34 , 73 ), we see no evidence that it can productively interact with either RPA–ssDNA or RAD51–ssDNA in our single molecule assays ( 35 ). It is important to recognize that BLM can only act upon the inactive ADP-bound form of the RAD51 filament ( 3 , 34 ), which is itself highly unstable ( 36 , 47 , 74 ), and BLM is unable to act upon the active ATP-bound form of the RAD51 filament ( 3 , 34 , 35 ). Indeed, we find that RPA and the active, ATP-bound form of RAD51 both block BLM interactions with ssDNA ( 35 ).…”

“…In these assays, we used either unlabeled RECQ5 and RPA–GFP or GFP–RECQ5 and unlabeled RPA. If unlabeled RECQ5 could unwind the dsDNA then we predicted this activity would be revealed by the binding of fluorescent RPA to the resulting ssDNA strands, as we have previously shown for yeast Sgs1 and human BLM helicases (Figure 9A ) ( 35 , 62 ). Assays with GFP–RECQ5 and unlabeled RPA cannot directly reveal unwinding activity, but they can be used to determine whether RECQ5 can bind to and translocate on dsDNA (Figure 9B ).…”

“…To directly examine RECQ5 antirecombinase activity at the single molecule level, we prepared RAD51–ssDNA curtains using unlabeled RAD51, as described previously (Figure 4A ) ( 35 , 39 , 50 ). Of note, we have previously shown that the resulting RAD51 filaments are extremely stable and do not spontaneously dissociate unless ATP and Ca 2+ are flushed from the sample chamber ( 47 ).…”

“…Our data demonstrate that RECQ5 exhibits robust translocase activity on RPA-, RAD51- and DMC1-bound ssDNA. Using double-stranded DNA curtains, we next sought to determine whether RECQ5 could translocate on lambda-DNA molecules (48 502-bp) that were tethered by both ends to the flow cell surface ( 35 , 61 ). In these assays, we used either unlabeled RECQ5 and RPA–GFP or GFP–RECQ5 and unlabeled RPA.…”

“…RECQ5 can disrupt RAD51–ssDNA filaments and in doing so acts as a negative regulator of RAD51 strand invasion activity ( 3 , 21 ). Interestingly, RECQ5 can dismantle active ATP-bound forms of the RAD51–ssDNA filament, whereas BLM can only dismantle the inactive ADP-bound form of RAD51 ( 3 , 25 , 34 , 35 ). This important difference suggests the possibility that RECQ5 may play a more prominent role than BLM in regulating RAD51 filament disassembly ( 3 ).…”