The RecA/RAD51 protein drives migration of Holliday junctions via polymerization on DNA

Abstract

The Holliday junction (HJ), a cross-shaped structure that physically links the two DNA helices, is a key intermediate in homologous recombination, DNA repair, and replication. Several helicase-like proteins are known to bind HJs and promote their branch migration (BM) by translocating along DNA at the expense of ATP hydrolysis. Surprisingly, the bacterial recombinase protein RecA and its eukaryotic homologue Rad51 also promote BM of HJs despite the fact they do not bind HJs preferentially and do not translocate along DNA. RecA/Rad51 plays a key role in DNA double-stranded break repair and homologous recombination. RecA/Rad51 binds to ssDNA and forms contiguous filaments that promote the search for homologous DNA sequences and DNA strand exchange. The mechanism of BM promoted by RecA/RAD51 is unknown. Here, we demonstrate that cycles of RecA/Rad51 polymerization and dissociation coupled with ATP hydrolysis drives the BM of HJs.H omologous recombination (HR) is the process responsible for maintaining genome stability in all living organisms; it is particularly important for repairing DNA double-strand breaks (1-5). The process of HR involves the enzymatic degradation of broken dsDNA ends into resected DNA duplex with protruding ssDNA tails (6, 7). Following resection, the central protein of HR, the prokaryotic (bacterial recombinase protein) RecA or its eukaryotic homolog Rad51, is loaded onto the ssDNA tails and forms a contiguous nucleoprotein filament (8, 9). Although RecA and Rad51 share only ∼30% sequence homology, the filaments they form and the conformational changes they induce in DNA are nearly identical (10).The RecA/Rad51-ssDNA filament possesses the unique ability to search for homologous dsDNA sequences and promote DNA strand exchange: an invasion of ssDNA into homologous DNA duplex that results in the displacement of the identical ssDNA from the duplex and formation of a joint molecule (JM) ( Fig. S1A) (11). Besides its essential DNA strand-exchange activity RecA/RAD51 recombinases have an ability to extend JMs by a kinetically distinct process known as heteroduplex extension or three-strand branch migration (BM), in which one DNA strand is progressively exchanged for another (11,12). When the extending heteroduplex reaches the ssDNA-dsDNA junction on the invading DNA strand, the 3-stranded JM is converted into a 4-stranded Holliday junction (HJ). Then, specialized DNA translocating proteins, like Escherichia coli RuvAB, bind to HJs and promote their migration by four-strand BM (otherwise referred to as the BM of HJs) (13). These DNA translocases are capable of bypassing sequence heterologies and disrupting nucleoprotein complexes encountered during BM. Finally, DNA repair synthesis by DNA polymerases takes place on JMs followed by their resolution.Surprisingly, the RecA/Rad51 recombinases can also promote four-strand BM (Fig. S1A) (14, 15). This activity may play a significant role in the initial stages of recombination in vivo by helping to form and stabilize HJs, and therefore it is important to u...

Mutant IDH1-Driven Cellular Transformation Increases RAD51-Mediated Homologous Recombination and Temozolomide Resistance

“…HR in turn requires the collective action of a variety of proteins including RAD51, which accumulates at the gamma-H2AX-marked sites of DNA double strand breaks and contributes to their repair (34, 35). To first determine if alterations in HR contributed to the ability of mutant IDH1-expressing cells to escape from TMZ-induced G2 arrest, we monitored the formation and disappearance of RAD51 foci in TMZ-treated E6/E7/hTERT cells lentivirally infected with a blank vector or a construct encoding WT or mutant IDH1.…”

Balancing self-renewal against genome preservation in stem cells: How do they manage to have the cake and eat it too?

“…The nucleoprotein complex of ssDNA-bound RAD51 is stabilized by RAD51 paralogues, which include five members in human, that is, XRCC2, XRCC3, RAD51B/RAD51L1, RAD51C/RAD51L2, and RAD51D/RAD51L3. RAD51 serves a key role in initiating strand invasion at the homologous sequence and driving the branch migration of the Holiday junction [150]. The mechanism for RAD51 recruitment following replication stalling is not entirely clear, but may involve breast cancer 2 (BRCA2) [151], SUMOylated BLM [152], SUMOylated RPA70 [153], RAD52 [154], and X-ray repair cross-complementing proteins 2 and 3 (XRCC2 and XRCC3) [155, 156].…”

Reconstituting the 4-Strand DNA Strand Exchange

“…Rad51 recombinases can promote extension of joint molecules via a kinetically and mechanistically distinct reaction, known as 3-strand branch migration. Experimental data indicate that branch migration is driven by cycles of recombinase dissociation, reassociation, and polymerization on the displaced ssDNA of joint molecules directed toward the ssDNA–dsDNA junction (Rossi, Mazina, Bugreev, & Mazin, 2011). Consequently, the branch migration activity depends on ATP hydrolysis which requires for dissociation of Rad51 recombinases from DNA (Kowalczykowski, 1991).…”

“…In contrast, the Rad51 DNA strand exchange activity does not require protein dissociation from DNA and can be carried out in the presence of nonhydrolyzable ATP analogs. The polarity of branch migration is determined by the direction of the recombinase polymerization on the displaced ssDNA strand; e.g., it is 5′–3′ for RecA and 3′–5′ for hRad51 (Konforti & Davis, 1992; Kowalczykowski, 1991; Rossi et al, 2011). …”

“…1). The described methodology can be applied for studies of branch migration promoted by Rad51 recombinases (Rossi et al, 2011), and also by helicase-like proteins, e.g., Rad54, BLM, or RecQ1 (Mazina et al, 2012). …”