Biochemistry of Human RecQ DNA Helicases and their Role in DNA Repair

Field: Biochemistry, Genetics and Molecular Biology (Life Sciences)

Proteins belonging to the RecQ DNA helicase family are highly conserved from bacteria to man and are implicated in removing aberrant DNA structures arising during DNA replication and repair to prevent inappropriate DNA recombination events. While E.coli and yeast have only one RecQ-type helicase, humans have five of these named RECQ1, BLM, WRN, RECQ4 and RECQ5. Inherited defects in the genes encoding for BLM, WRN and RECQ4 have been found to give rise to Bloomʼs, Wernerʼs and Rothmund-Thomson syndrome, respectively. These severe recessive disorders are associated with genomic instability, cancer predisposition and premature aging. Association of inherited defects in the RECQ1 and RECQ5 genes with human disease has not been reported. However, knockout studies in chicken DT40 cells and in mice suggested that these genes function as tumor suppressors through enforcing chromosomal stability. A numerous biochemical and cellular studies indicated that the multiple RecQ homologues in human cells have non-redundant biological roles. However, the exact DNA transactions mediated by these proteins remain elusive. \nThe goal of this thesis was to advance our understanding of the cellular functions of RECQ5 and WRN helicases. In the first project (Kanagaraj et al., 2006), we explored the possible role for RECQ5 in replication fork management. Earlier biochemical studies from our group have shown that RECQ5 functions as a 3ʼ-5ʼ DNA helicase and it has also an ability to promote DNA strand annealing. We analyzed the action of RECQ5 on various synthetic forked DNA structures containing either heterologous or homologous arms. We found that the mode of action of RECQ5 helicase on these structures was different as compared to other human RecQ helicases. For example, we found that on forked structures with heterologous arms, RECQ5 showed preference for unwinding of the lagging-strand arm, whereas BLM and WRN helicases displayed a strong preference for unwinding of the parental arm. We also showed that RECQ5 promoted strand exchange between homologous arms of a synthetic forked DNA structure in a reaction dependent on ATP hydrolysis and that human replication protein A (hRPA) stimulated this reaction. Further, we identified a domain located in the non-conserved C- terminal portion of RECQ5 (a region spanning amino acids 561-651) as being important for its ability to unwind the lagging-strand arm of the fork and to promote strand exchange on hRPA-coated forked structures. Our cellular studies revealed that RECQ5 was associated with the DNA replication factories in S phase nuclei and persisted at the sites of stalled replication forks after exposure of cells to UV irradiation and cisplatin. Moreover, RECQ5 was found to physically interact with the polymerase processivity factor proliferating cell nuclear antigen in vitro and in vivo. Taken together, our findings suggested that RECQ5 might be involved in the reactivation of stalled replication forks. \nIn contrast to the other human RecQ homologues, RECQ5 exists in at least three different isoforms (named RECQ5α, RECQ5β and RECQ5γ) that result from alternative mRNA splicing. Although the RECQ5β isoform has been biochemically well characterized, no information was available for the other two isoforms. In the second project, we studied the biochemical properties of the two uncharacterized isoforms of RECQ5 in collaboration with Dr. Xiʼs laboratory (Ren et al., 2008). This work clarified the function of the zinc (Zn2+)-binding motif that is present in the RECQ5β protein but not in the other two short isoforms. Our initial studies have shown that the short RECQ5 isoforms, RECQ5α and RECQ5γ, containing all conserved helicase motifs do not possess ATPase and DNA unwinding activities. Additional biochemical experiments with the RECQ5α protein revealed that this RECQ5 isoform was proficient in DNA strand annealing. Surprisingly, a fragment of RECQ5β (1-475 aa) containing an intact Zn2+- binding motif did not exhibit any strand-annealing activity and was proficient in DNA unwinding. Further, our results demonstrated that Zn2+-binding motif of the human RECQ5β protein negatively regulates the intrinsic DNA strand-annealing activity of the helicase domain and is essential for the ATPase and helicase activities of the enzyme. Moreover, quantitative kinetic measurements indicated that the regulatory role of the Zn2+-binding motif was achieved by enhancing the DNA binding affinity of the enzyme. Taking into consideration that Zn2+-binding motif is highly conserved among RecQ family DNA helicases, we believe that the novel intra-molecular regulation of RECQ5 catalytic activity mediated by the Zn2+-binding motif (identified in this work) may represent a universal regulation mode for all RecQ family helicases. To further explore the biological role of the human RECQ5 helicase, we employed a proteomic approach to identify proteins associated with RECQ5 in vivo. One of the RECQ5 binding partners we found in this analysis was the MRE11/RAD50/NBS1 (MRN) complex, a nuclease that functions in many aspects of DNA metabolism including DNA double-strand breaks (DSBs). Accumulating evidence suggest that the MRN nuclease acts as a DSB sensor and activates the ATM-signalling pathway. Accordingly, defects in the genes encoding for the components of the MRN complex lead to severe DNA damage sensitivity, high genomic instability, shortening of telomeres and aberrant meiosis. In the third project (Zheng et al., manuscript to be submitted), we investigated the biological significance of the association of RECQ5 with the MRN complex. We have demonstrated that RECQ5 binds directly to the MRE11 and NBS1 subunits of the MRN complex. The MRN complex and RECQ5 were found to co-localize at sites of stalled DNA replication forks and sites of DNA DSBs. Further, we showed that the MRN complex is required for the recruitment of RECQ5 to the sites of DNA damage. In addition, our biochemical experiments revealed that RECQ5 negatively regulates the MRN complex by attenuating its 3ʼ-5ʼ exonuclease activity. Collectively, these data establish a functional relationship between RECQ5 and the MRN complex in the cellular response to DNA damage. \nIn the last project, we characterized the interaction between the Werner syndrome protein (WRN) and the mismatch repair (MMR) initiation factors (Saydam et al., 2007). The MMR system maintains genomic integrity by correcting DNA replication errors and preventing recombination between divergent sequences. Werner syndrome (WS) is a severe recessive disorder characterized by premature aging, cancer predisposition and genomic instability. The gene mutated in WS encodes a bi-functional enzyme called WRN that acts as a 3ʼ-5ʼ DNA helicase and a 3 -5 exonuclease, but its exact role in DNA metabolism is poorly understood. We found that WRN physically interacts with the MMR initiation factors, MutSα (heterodimer of MSH2/MSH6), MutSβ (heterodimer of MSH2/MSH3) and MutLα (heterodimer of MLH1/PMS2). Also, we found that the helicase activity of WRN on forked DNA structures containing a 3ʼ-single-stranded arm was strongly stimulated by MutSα or MutSβ, but not by MutLα. Moreover, the stimulatory effect of MutSα on WRN-mediated unwinding was enhanced by presence of a G/T mismatch in the DNA duplex ahead of the fork. Collectively, these data are consistent with results of genetic experiments conducted in yeast suggesting that MMR factors act in conjunction with a RecQ-type helicase to reject recombination between divergent sequences. \n \n \nProteine, die zur RecQ DNA Helikase Familie gehören, sind hoch konserviert vom Bakterium bis hin zum Menschen. RecQ Helikasen werden zum Entfernen von ungewöhnlicher DNA Strukturen benötigt, die während der DNA Replikation und Reparatur entstehen und verhindern daher eine übermässige DNA Rekombination (Bachrati & Hickson, 2003). Während E.coli und Hefe nur eine RecQ Helikase haben, gibt es beim Menschen fünf Mitglieder dieser Proteine, nämlich RECQ1, BLM, WRN, RECQ4 und RECQ5. Vererbte Defekte im BLM, WRN oder RECQ4 Gen werden in Patienten mit Bloom, Werner oder Rothmund-Thomson Syndrom gefunden. Dies sind schwere rezessive Erkrankungen, die mit genomischer Instabilität, Krebsprädisposition und beschleunigtem Alterungsprozess assoziiert sind. Vererbte Defekte im RECQ1 und RECQ5 Gen sind bis jetzt nicht in Verbindung gebracht worden mit Erkrankungen beim Menschen. Studien mit aviären DT40 knock-out Zellen und in Mäusen deuten jedoch darauf hin, dass diese Gene auch als Tumorsuppressoren agieren und die chromosomale Stabilität erhöhen. Eine Anzahl biochemischer und zellulärer Studien zeigen auf, dass die mehrfachen RecQ Homologe in der humanen Zelle nicht redundante biologische Funktionen haben. Die exakte DNA Transaktion, die von diesen Proteinen vermittelt wird, bleibt hingegen ungewiss. \nDas Ziel dieser Dissertation war die zelluläre Funktion der RECQ5 und WRN Helikase besser zu definieren. Im ersten Projekt (Kanagaraj et al., 2006), untersuchten wir die mögliche Funktion von RECQ5 an der Replikationsgabel. Frühere biochemische Studien in unserer Gruppe haben gezeigt, dass RECQ5 eine 3ʼ-5ʼ DNA Helikase ist und das Zusammenlegen von einzelsträngiger DNA beschleunigen kann. Wir analysierten die Aktivität von RECQ5 mit verschiedenen synthetischen gabelförmiger DNA Strukturen, die entweder heterologe oder homologe Arme hatten. Wir fanden andere Aktivitätsmuster bei RECQ5 als bei anderen humanen RecQ Helikasen. Zum Beispiel zeigte RECQ5 eine Präferenz den Folgestrang zu entwinden, bei einer gabelförmiger Duplex-DNA mit heterologen Armen, während BLM und WRN Helikasen eine strarke Präferenz aufwiesen den parentalen Arm zu entwinden. Wir zeigten auch, dass RECQ5 eine Strangaustausch-Reaktion zwischen den homologen Armen einer synthetischen gabelförmigen DNA Struktur katalysiert. Diese Reaktion war abhängig von ATP Hydrolyse und wurde stimuliert d