Biochemical Characterization of AtRECQ3 Reveals Significant Differences Relative to Other RecQ Helicases

Abstract

Members of the conserved RecQ helicase family are important for the preservation of genomic stability. Multiple RecQ homologs within one organism raise the question of functional specialization. Whereas five different homologs are present in humans, the model plant Arabidopsis (Arabidopsis thaliana) carries seven RecQ homologs in its genome. We performed biochemical analysis of AtRECQ3, expanded upon a previous analysis of AtRECQ2, and compared their properties. Both proteins differ in their domain composition. Our analysis demonstrates that they are 3# to 5# helicases with similar activities on partial duplex DNA. However, they promote different outcomes with synthetic DNA structures that mimic Holliday junctions or a replication fork. AtRECQ2 catalyzes Holliday junction branch migration and replication fork regression, while AtRECQ3 cannot act on intact Holliday junctions. The observed reaction of AtRECQ3 on the replication fork is in line with unwinding the lagging strand. On nicked Holliday junctions, which have not been intensively studied with RecQ helicases before, AtRECQ3, but not AtRECQ2, shows a clear preference for one unwinding mechanism. In addition, AtRECQ3 is much more efficient at catalyzing DNA strand annealing. Thus, AtRECQ2 and AtRECQ3 are likely to perform different tasks in the cell, and AtRECQ3 differs in its biochemical properties from all other eukaryotic RECQ helicases characterized so far.RecQ helicases are important players in the maintenance of genomic stability in prokaryotes and eukaryotes (for reviews covering several aspects of RecQ helicases, see

Fork sensing and strand switching control antagonistic activities of RecQ helicases

“…Fast rezipping events as seen for AtRECQ2 were never observed. When using AtRECQ3 K64M8 with a mutation in the Walker A motif, no unwinding was detected. AtRECQ3 was also by far not able to unwind the full hairpin.…”

“…8). For this, we use single-molecule measurements that have in recent years become a valuable complement to X-ray crystallography and classical biochemical methodology in mechanistic investigations of helicases.…”

Genome-wide analysis of helicase gene family from rice and Arabidopsis: a comparison with yeast and human

“…In Arabidopsis, RecQ2 catalyzes Holliday junction branch migration and replication fork regression, while RecQ3 can not act on intact Holliday junctions thus suggesting that these RecQ helicases perform different functions in the cell (Kobbe et al 2009). Moreover, it was also shown that AtRecQ3 differs in its biochemical properties from all other eukaryotic RecQ helicases characterized so far (Kobbe et al 2009). …”

RAD5A, RECQ4A, and MUS81 Have Specific Functions in Homologous Recombination and Define Different Pathways of DNA Repair inArabidopsis thaliana

“…Homologs of both proteins have been shown to regress replication forks (Kanagaraj et al, 2006;Machwe et al, 2006;Ralf et al, 2006;Blastyá k et al, 2007;Kobbe et al, 2009). Both single mutant lines showed a high sensitivity to DNA-damaging agents that can cause replication fork stalling.…”

“…The minor reduction of HR efficiency detected with the SSA substrate might well be due to lack of the RECQ4A function during resection in our experimental system. Alternatively, RECQ4A might also be involved in reannealing the single-strand ends because some RecQ helicases have been shown to possess annealing activity (Garcia et al, 2004;Cheok et al, 2005;Sharma et al, 2005;Kobbe et al, 2009;Chen and Brill, 2010).…”