Mechanisms by Which Bloom Protein Can Disrupt Recombination Intermediates of Okazaki Fragment Maturation

Abstract

Bloom syndrome is a familial genetic disorder associated with sunlight sensitivity and a high predisposition to cancers. The mutated gene, Bloom protein (BLM), encodes a DNA helicase that functions in genome maintenance via roles in recombination repair and resolution of recombination structures. We designed substrates representing illegitimate recombination intermediates formed when a displaced DNA flap generated during maturation of Okazaki fragments escapes cleavage by flap endonuclease-1 and anneals to a complementary ectopic DNA site. Results show that displaced, replication protein A (RPA)-coated flaps could readily bind and ligate at the complementary site to initiate recombination. RPA also displayed a strand-annealing activity that hastens the rate of recombination intermediate formation. BLM helicase activity could directly disrupt annealing at the ectopic site and promote flap endonuclease-1 cleavage. Additionally, BLM has its own strand-annealing and strand-exchange activities. RPA inhibited the BLM strand-annealing activity, thereby promoting helicase activity and complex dissolution. BLM strand exchange could readily dissociate invading flaps, e.g. in a D-loop, if the exchange step did not involve annealing of RPA-coated strands. Use of ATP to activate the helicase function did not aid flap displacement by exchange, suggesting that this is a helicase-independent mechanism of complex dissociation. When RPA could bind, it displayed its own strand-exchange activity. We interpret these results to explain how BLM is well equipped to deal with alternative recombination intermediate structures. Bloom protein (BLM)2 is a member of the RecQ family of 3Ј-5Ј helicases that assist in maintaining genome stability. Mutation or loss of function of the BLM protein causes Bloom syndrome (BS), an autosomal recessive disease characterized by sunlight sensitivity, proportional dwarfism, and a high predisposition toward many different types of cancer (1). Cells with BLM deficiency show increased chromosomal abnormalities, including hyper-recombination, elevated rates of sister chromatid exchange, and the abnormal accumulation of replication intermediates, resulting in an increase in the overall level of genomic instability (2-4). Knock-out of BLM in mice causes embryonic lethality, whereas some mutations produce live mice prone to tumorigenesis (5, 6).BLM plays a role in several critical genome maintenance pathways. Immunodepletion of Xenopus BLM inhibits the replication of DNA in reconstituted nuclei, suggesting that BLM is directly involved in DNA replication (7). Telomere proteins TRF2 and TRF1 colocalize with BLM in immortalized cells lines and regulate its helicase activity in vivo, signifying a role for BLM in telomere maintenance (8). BLM assists in the recovery of stalled replication forks and in the prevention of repeat expansion by stabilizing repeated sequences (9 -13). Additionally, BLM has been proposed to promote proper intermediate resolution and suppress crossovers in the homologous recombination pathwa...

Catalysis of Strand Annealing by Replication Protein A Derives from Its Strand Melting Properties

“…Instead, we found that RPA could promote strand annealing and subsequent recombination (34). Our current results show that RPA-coated flaps of a variety of lengths could be precursors to recombination, suggesting a need for Dna2 helicase to eliminate these flaps before recombination occurs.…”

“…It has been hypothesized that some flaps become long enough to be coated by RPA (30,32,33). When we tested flap substrates with RPA-coated flaps, we were surprised to find that the RPA promoted annealing of the flap to other single-stranded template DNA (34). This result suggested that RPA alone can support rather than suppress recombination.…”

“…We previously acquired evidence that RPA can accelerate the annealing of DNA strands (34). Our current work is an investigation of the conditions and substrate structures that support RPA-catalyzed strand annealing.…”

“…We previously analyzed the propensity of single-stranded flaps created during Okazaki fragment maturation to participate in illegitimate recombination (34). Eukaryotic Okazaki fragment processing has been proposed to occur via two pathways.…”

The MCM and RecQ Helicase Families: Ancient Roles in DNA Replication and Genomic Stability Lead to Distinct Roles in Human Disease

“…In vitro studies with use of substrates similar to Okazaki fragments showed BLM stimulation of flap endonuclease [105] (a protein that functions in lagging strand synthesis [106]). BLM functions to prevent the association of homologous sequences in the displaced flap DNA of the Okazaki fragment and the sister chromatid [107][108][109][110]. D-loops are formed when a ssDNA tail invades a homologous duplex, and BLM has a preference for dissociating D-loops with a 5' invaded end suggesting a selection of recombination intermediates that are not extended by polymerase [93,111,112].…”