Role for the fission yeast RecQ helicase in DNA repair in G2.

Members of the RecQ helicase subfamily are mutated in several human genomic instability syndromes, such as Bloom, Werner, and Rothmund-Thomson syndromes. We show that Rqh1, the single Schizosaccharomyces pombe homologue, is a 3'-to-5' helicase and exists with Top3 in a high-molecular-weight complex. top3 deletion is inviable, and this is suppressed by concomitant loss of rqh1 helicase activity or loss of recombination functions. This is consistent with RecQ helicases in other systems. By using epistasis analysis of the UV radiation sensitivity and by analyzing the kinetics of Rhp51 (Rad51 homologue), Rqh1, and Top3 focus formation in response to UV in synchronized cells, we identify the first evidence of a function for Rqh1 and Top3 in the repair of UV-induced DNA damage in G(2). Our data provide evidence that Rqh1 functions after Rad51 focus formation during DNA repair. We also identify a function for Rqh1 upstream of recombination in an Rhp18-dependent (Rad18 homologue) pathway. The model that these data allow us to propose helps to reconcile different interpretations of RecQ family helicase function that have arisen between work based on the S. pombe system and models based on studies of Saccharomyces cerevisiae SGS1 suggesting that RecQ helicases act before Rad51

Dissecting Inter-domain Cooperativity in the Folding of a Multi Domain Protein.

Correct protein folding underlies all cellular functions. While there are detailed descriptions and a good understanding of protein folding pathways for single globular domains there is a paucity of quantitative data regarding folding of multidomain proteins. We have here investigated the folding of a three-domain supramodule from the protein PSD-95, consisting of one PDZ domain, one SH3 domain and one guanylate kinase-like (GK) domain. This supramodule has previously been shown to work as one functional unit with regard to ligand binding. We used equilibrium and kinetic folding experiments to demonstrate that the PDZ domain folds faster and independently from the SH3-GK tandem, which folds as one cooperative unit. However, concurrent folding of the PDZ domain slows down folding of SH3-GK by non-native interactions, resulting in an off-pathway folding intermediate. Our data contribute to an emerging description of multidomain protein folding in which individual domains cannot a priori be viewed as separate folding units

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