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

Abstract

RECQ5 is one of five RecQ helicases found in humans and is thought to participate in homologous DNA recombination by acting as a negative regulator of the recombinase protein RAD51. Here, we use kinetic and single molecule imaging methods to monitor RECQ5 behavior on various nucleoprotein complexes. Our data demonstrate that RECQ5 can act as an ATP-dependent single-stranded DNA (ssDNA) motor protein and can translocate on ssDNA that is bound by replication protein A (RPA). RECQ5 can also translocate on RAD51-coated ssDNA and readily dismantles RAD51–ssDNA filaments. RECQ5 interacts with RAD51 through protein–protein contacts, and disruption of this interface through a RECQ5–F666A mutation reduces translocation velocity by ∼50%. However, RECQ5 readily removes the ATP hydrolysis-deficient mutant RAD51–K133R from ssDNA, suggesting that filament disruption is not coupled to the RAD51 ATP hydrolysis cycle. RECQ5 also readily removes RAD51–I287T, a RAD51 mutant with enhanced ssDNA-binding activity, from ssDNA. Surprisingly, RECQ5 can bind to double-stranded DNA (dsDNA), but it is unable to translocate. Similarly, RECQ5 cannot dismantle RAD51-bound heteroduplex joint molecules. Our results suggest that the roles of RECQ5 in genome maintenance may be regulated in part at the level of substrate specificity.

Mechanistic Insights From Single-Molecule Studies of Repair of Double Strand Breaks

“…Though similar to the case of Srs2, Sgs1 action was also inhibited by Dmc1 (Crickard et al, 2019). Functional conservation of RECQ helicases in anti-recombination was recently demonstrated for the human RECQ5 on DNA curtain (Figure 4D, left) (Xue et al, 2021). RECQ5 not only translocated on ssDNA bound by RPA, RAD51, or DMC1, but also removed these proteins in the process (Figure 4D, right).…”

Srs2 and Pif1 as Model Systems for Understanding Sf1a and Sf1b Helicase Structure and Function

“…One of the most well characterized roles of Srs2 is its ability to regulate HR by removing the Rad51 recombinase from ssDNA recombination intermediates ( Figure 4 A) [ 1 , 6 , 71 , 72 ]. This type of “antirecombinase” activity has now also been demonstrated for several other helicases, including E. coli UvrD [ 61 ], S. cerevisiae Sgs1 [ 73 ], and the human helicases FBH1, PARI, BLM, RECQ5 [ 74 , 75 , 76 , 77 , 78 , 79 , 80 ]. As indicated above, several genetic studies revealed that Srs2 played a crucial role in constraining HR, and the mechanisms by which Srs2 accomplished this task was revealed in two biochemical studies which demonstrated that purified recombinant Srs2 could physically strip Rad51 from ssDNA [ 71 , 72 ].…”

DNA bridges: A novel platform for single-molecule sequencing and other DNA-protein interaction applications

“…Additional applications for this platform can be envisioned in protein-nucleic acid interaction studies [ 44 – 47 ] ( S3 Fig ). For instance, studies that examine the cooperative binding of multisubunit transcription factors would allow each of the proteins under investigation to be added individually to the system while being monitored by optical readout.…”

“…Additional applications for this platform can be envisioned in protein-nucleic acid interaction studies [44][45][46][47] (S3 Fig) . For instance, studies that examine the cooperative binding of multisubunit transcription factors would allow each of the proteins under investigation to be added individually to the system while being monitored by optical readout. Tagged enzymes such as DNA and RNA polymerases could be used to record template engagement in realtime, and kinetic data could be extracted from observations of their movement.…”