Abstract
There are lines of evidence that the Bloom syndrome helicase, BLM, catalyzes regression of stalled replication forks and disrupts displacement loops (D-loops) formed during homologous recombination (HR). Here we constructed a forked DNA with a 3′ single-stranded gap and a 5′ double-stranded handle to partly mimic a stalled DNA fork and used magnetic tweezers to study BLM-catalyzed unwinding of the forked DNA. We have directly observed that the BLM helicase may slide on the opposite strand for some distance after duplex unwinding at different forces. For DNA construct with a long hairpin, progressive unwinding of the hairpin is frequently interrupted by strand switching and backward sliding of the enzyme. Quantitative study of the uninterrupted unwinding length (time) has revealed a two-state-transition mechanism for strand-switching during the unwinding process. Mutational studies revealed that the RQC domain plays an important role in stabilizing the helicase/DNA interaction during both DNA unwinding and backward sliding of BLM. Especially, Lys1125 in the RQC domain, a highly conserved amino acid among RecQ helicases, may be involved in the backward sliding activity. We have also directly observed the in vitro pathway that BLM disrupts the mimic stalled replication fork. These results may shed new light on the mechanisms for BLM in DNA repair and homologous recombination.
Highlights
The RecQ family helicases have been highly conserved during evolution from bacteria to human
If the force is around 14 pN, the hairpin hops between the folded and unfolded states (Supplementary Figure S1). It is by this way that we check if the magnetic bead is tethered to the surface through a single DNA construct or not
The low processivity of BLM may arise from its high ssDNA release rate in the adenosine diphosphate (ADP) state [5,42]
Summary
The RecQ family helicases have been highly conserved during evolution from bacteria to human. BLM is a DNA structure-specific helicase that unwinds DNA in 3 -5 direction [4,5], and shows an apparent preference for substrates like Holliday junctions, G-quadruplexes, DNA displacement loops (D-loops) and stalled replication forks [6,7,8,9,10,11,12]. These substrates represent different DNA structures that can be formed in vivo during DNA replication and homologous recombination (HR). It has become evident in recent years that HR and repair of stalled replication forks are intimately connected and, in many cases, loss of function of a helicase can have an adverse effect on both HR and replication fork management [13,14,15,16]
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