The motor activity of DNA2 functions as an ssDNA translocase to promote DNA end resection

DNA2 nuclease–helicase functions in DNA replication and recombination. This requires the nuclease of DNA2, while, in contrast, the role of the helicase activity has been unclear. We now show that the motor activity of both recombinant yeast and human DNA2 promotes efficient degradation of long stretches of ssDNA, particularly in the presence of the replication protein A. This degradation is further stimulated by a direct interaction with a cognate RecQ family helicase, which functions with DNA2 in DNA end resection to initiate homologous recombination. Consequently, helicase- deficient yeast dna2 K1080E cells display reduced resection speed of HO-induced DNA double-strand breaks. These results support a model of DNA2 and the RecQ family helicase partner forming a bidirectional motor machine, where the RecQ family helicase is the lead helicase, and the motor of DNA2 functions as a ssDNA translocase to promote degradation of 5'-terminated DNA

Biochemical Analysis and In Vivo Role of Dna2 Nuclease-Helicase

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

Sumoylation regulates the stability and nuclease activity of Saccharomyces cerevisiae Dna2

Dna2 is an essential nuclease-helicase that acts in several distinct DNA metabolic pathways including DNA replication and recombination. To balance these functions and prevent unscheduled DNA degradation, Dna2 activities must be regulated. Here we show that Saccharomyces cerevisiae Dna2 function is controlled by sumoylation. We map the sumoylation sites to the N-terminal regulatory domain of Dna2 and show that in vitro sumoylation of recombinant Dna2 impairs its nuclease but not helicase activity. In cells, the total levels of the non-sumoylatable Dna2 variant are elevated. However, non-sumoylatable Dna2 shows impaired nuclear localization and reduced recruitment to foci upon DNA damage. Non-sumoylatable Dna2 reduces the rate of DNA end resection, as well as impedes cell growth and cell cycle progression through S phase. Taken together, these findings show that in addition to Dna2 phosphorylation described previously, Dna2 sumoylation is required for the homeostasis of the Dna2 protein function to promote genome stability

Sumoylation regulates the stability and nuclease activity of Saccharomyces cerevisiae Dna2

Dna2 is an essential nuclease-helicase that acts in several distinct DNA metabolic pathways including DNA replication and recombination. To balance these functions and prevent unscheduled DNA degradation, Dna2 activities must be regulated. Here we show that Saccharomyces cerevisiae Dna2 function is controlled by sumoylation. We map the sumoylation sites to the N-terminal regulatory domain of Dna2 and show that in vitro sumoylation of recombinant Dna2 impairs its nuclease but not helicase activity. In cells, the total levels of the non-sumoylatable Dna2 variant are elevated. However, non-sumoylatable Dna2 shows impaired nuclear localization and reduced recruitment to foci upon DNA damage. Non-sumoylatable Dna2 reduces the rate of DNA end resection, as well as impedes cell growth and cell cycle progression through S phase. Taken together, these findings show that in addition to Dna2 phosphorylation described previously, Dna2 sumoylation is required for the homeostasis of the Dna2 protein function to promote genome stability

DNA2 drives processing and restart of reversed replication forks in human cells

Accurate processing of stalled or damaged DNA replication forks is paramount to genomic integrity and recent work points to replication fork reversal and restart as a central mechanism to ensuring high-fidelity DNA replication. Here, we identify a novel DNA2- and WRN-dependent mechanism of reversed replication fork processing and restart after prolonged genotoxic stress. The human DNA2 nuclease and WRN ATPase activities functionally interact to degrade reversed replication forks with a 5'-to-3' polarity and promote replication restart, thus preventing aberrant processing of unresolved replication intermediates. Unexpectedly, EXO1, MRE11, and CtIP are not involved in the same mechanism of reversed fork processing, whereas human RECQ1 limits DNA2 activity by preventing extensive nascent strand degradation. RAD51 depletion antagonizes this mechanism, presumably by preventing reversed fork formation. These studies define a new mechanism for maintaining genome integrity tightly controlled by specific nucleolytic activities and central homologous recombination factors

Replication intermediates that escape Dna2 activity are processed by Holliday junction resolvase Yen1

Cells have evolved mechanisms to protect, restart and repair perturbed replication forks, allowing full genome duplication, even under replication stress. Interrogating the interplay between nuclease-helicase Dna2 and Holliday junction (HJ) resolvase Yen1, we find the Dna2 helicase activity acts parallel to homologous recombination (HR) in promoting DNA replication and chromosome detachment at mitosis after replication fork stalling. Yen1, but not the HJ resolvases Slx1-Slx4 and Mus81-Mms4, safeguards chromosome segregation by removing replication intermediates that escape Dna2. Post-replicative DNA damage checkpoint activation in Dna2 helicase-defective cells causes terminal G2/M arrest by precluding Yen1-dependent repair, whose activation requires progression into anaphase. These findings explain the exquisite replication stress sensitivity of Dna2 helicase-defective cells, and identify a non-canonical role for Yen1 in the processing of replication intermediates that is distinct from HJ resolution. The involvement of Dna2 helicase activity in completing replication may have implications for DNA2-associated pathologies, including cancer and Seckel syndrome

The motor activity of DNA2 functions as an ssDNA translocase to promote DNA end resection

DNA2 nuclease-helicase functions in DNA replication and recombination. This requires the nuclease of DNA2, while, in contrast, the role of the helicase activity has been unclear. We now show that the motor activity of both recombinant yeast and human DNA2 promotes efficient degradation of long stretches of ssDNA, particularly in the presence of the replication protein A. This degradation is further stimulated by a direct interaction with a cognate RecQ family helicase, which functions with DNA2 in DNA end resection to initiate homologous recombination. Consequently, helicase-deficient yeast dna2 K1080E cells display reduced resection speed of HO-induced DNA double-strand breaks. These results support a model of DNA2 and the RecQ family helicase partner forming a bidirectional motor machine, where the RecQ family helicase is the lead helicase, and the motor of DNA2 functions as a ssDNA translocase to promote degradation of 5'-terminated DNA