Biochemical Analysis and In Vivo Role of Dna2 Nuclease-Helicase

Abstract

Genome instability is a characteristic of almost all human cancers and is a prerequisite for acquisition of further hallmarks of cancer. While in hereditary cancers it arises due to mutations in DNA repair genes, in sporadic cancers it appears that oncogene-induced replication stress is the main cause for genomic instability. Hence, faithful DNA replication and repair are crucial to preserve genome integrity, thus contributing to cancer prevention, and to properly transmit genetic information across generations. Dna2 is an essential enzyme that is conserved from yeast to humans and is involved in the maintenance of genome stability at multiple levels. It plays a role in unperturbed DNA replication as well as under conditions of replication stress. In addition, Dna2 functions together with Sgs1 (in yeast; Bloom or Werner in humans) in the repair of genotoxic double-strand DNA breaks, specifically in DNA end resection, which is the commitment step to mostly error-free homologous recombination pathway. Furthermore, Dna2 was described to be part of telomeric and mitochondrial DNA maintenance systems, and to mediate checkpoint activation in yeast. During my PhD I was investigating the functions of Dna2 in Saccaromyces cerevisiae and was mainly working with purified yeast proteins. First, we expressed and purified yeast Dna2 and were able to show that it possesses not only a nuclease, but also a vigorous helicase activity. Then we set out to analyze the regulation of the two activities within the Dna2 protein. Using in vitro and in vivo approaches we show that yeast Dna2 is regulated by a post-translational modification termed sumoylation. On the biochemical level, sumoylation of the N-terminus of Dna2 selectively attenuated its nuclease activity, thus changing the balance between the helicase and the nuclease within the protein. In vivo, we show that sumoylation of Dna2 is increased in the late S/G2 phases of the cell cycle and appears to be involved in regulation of Dna2 upon treatment with alkylating agents. Next, we addressed the essential function of Dna2 in lagging strand DNA replication, where it acts together with Fen1 (Flap endonuclease I) in the processing of long DNA flap structures arising during the maturation of Okazaki fragments. The nucleolytic cleavage of these flaps is required for removal of the potentially mutagenic RNA/DNA primer initially used for the synthesis of the Okazaki fragment and allows ligation of the neighboring fragments. While short flaps are processed by Fen1, long flaps that are bound by replication protein A (RPA) need sequential cleavage by both Dna2 and Fen1 enzymes. Using in vitro reconstitution assays, we show that Dna2 is capable of processing the long flaps to products that can be subsequently ligated by DNA ligase I and that Dna2 is highly efficient as a sole nuclease in Okazaki fragment maturation in concert with replication, without the requirement of a second nucleolytic activity of Fen1. We suggest that Fen1 processes most of the flaps in S phase, where it is mainly expressed, and Dna2 is responsible for the cleavage of DNA flaps at later replication time points or possibly also during post-replicative repair processes. Furthermore, we examined the role of Dna2 motor activity in the context of DNA end resection, which initiates homologous recombination. Employing biochemical approaches we show that on long stretches of ssDNA the motor activity of Dna2 acts as a ssDNA translocase, especially in presence of RPA, and highly stimulates efficient DNA degradation, an effect that we also see when it acts together with Sgs1. We propose that in resection the motor activity of Dna2 functions as a ssDNA translocase, rather than a helicase, and is thus allowing Dna2 to keep up with Sgs1 and promoting efficient DNA degradation. Moreover, in collaborative projects we were able to show that Dna2 is also involved in the processing of replication forks that reversed upon replication stress and provide further evidence that human DNA2 cooperates with BLM and WRN to promote long-range resection. Additionally, another collaboration yielded proof that the helicase activity of Dna2 is required for the response to replication stress and for the completion of replication. Lastly, single-molecule analysis of RPA association to forked DNA substrates done by our collaborators sheds light on its mechanistic role during DNA replication.