Rothmund Thomson Syndromes (RTS) is a rare disease associated with genome instability, predisposition to cancer, skin and skeletal abnormalities, and some features of premature aging. The disease is caused by mutation in RECQL4, a putative helicase of the RecQ family that also includes WRN and BLM, proteins defective in Werner and Bloom syndromes, respectively. To understand the mechanism of this disease, we have purified a human RECQL4 complex and identified its associated components. We found that the bulk of RECQL4 was present in cytoplasmic extracts of HeLa cells, in contrast to the largely nuclear BLM and WRN helicases. However, in untransformed WI-38 fibroblasts, RECQL4 was found to be largely nuclear, and was present at significantly lower total levels than in transformed HeLa cells. RECQL4 from HeLa cells was isolated as a stable complex with UBR1 and UBR2. These 200 kD proteins are ubiquitin ligases of the N-end rule pathway, whose substrates include proteins with destabilizing N-terminal residues. The functions of this proteolytic pathway include the regulation of peptide import, chromosome stability, meiosis, apoptosis, and cardiovascular development. Although the known role of UBR1 and UBR2 is to mediate polyubiquitylation (and subsequent degradation) of their substrates, the UBR1/2-bound RECQL4 was not ubiquitylated in vivo, and was a long-lived protein in HeLa cells. The isolated RECQL4-UBR1/2 complex had a DNA-stimulated ATPase activity but was inactive in DNA-based assays for helicases and translocases, the assays in which the BLM helicase was active. Our data suggest that RECQL4 and ubiquitin ligases of the N-end rule pathway may play a role in maintaining genomic stability. We have also started purification and characterization of RECQL5, another RecQ helicase involved in maintaining genome stability in mammalian cells. Mutation of this gene has been shown to result in genomic instability in cells. To investigate how RecQL5 helps to maintain genome stability, we performed unbiased purification of the RecQL5-associated complex. Surprisingly, we found that RecQL5 associated with RNA polymerase II (Pol II). While our project is in progress, two other labs have independently reported that RecQL5 associates with Pol II. RecQL5 has also been reported to interact with TopoIIIa, TopoIIIb, Rad51, and PCNA. However, it remains unclear which of these interactions are important for RecQL5 in genome stabilization. It is also unknown whether its helicase activity is important for RecQL5 to function in vivo. We obtained RecQL5-/- DT40 cells from Dr. Enomotos group, and used them to investigate which activity of RecQL5, its interaction with Pol II or its helicase activity, is important for maintaining genome stability. We first mapped the domain in RecQL5 that interacts with Pol II. The results show that a 100 amino acid non-conserved region outside of the helicase domain of RecQL5 is both necessary and sufficient for interaction with Pol II. We then made several point mutants in this conserved region, and were able to identify a single point mutant that disrupts the interaction of RecQL5 and pol II. RecQL5-/- DT40 cells transfected by this mutant display SCE level and camptothecin sensitivity significantly higher than those from cells transfected with wildtype RecQL5, indicating that the interaction with Pol II is important for RecQL5 to maintain genome stability. To further investigate whether helicase activity of RecQL5 is important, we generated a helicase domain deletion mutant and a single point mutant that inactivate its helicase activity. While both mutants retained the ability to interact with Pol II, they fail to fully suppress the higher SCE frequency and correct increased camptothecin sensitivity of the RecQL5-/- DT40 cells, indicating that the helicase activity of RecQL5 is also important for genome maintenance. We noticed that introduction of either the Pol II-interaction mutant or the helicase point mutant did partially correct the SCE and camptothecin phenotypes of RecQL5-/- cells, even though such correction was incomplete compared to the wildtype protein, indicating that disruption of either activity does not fully inactivate RecQL5. The data raised the conjecture that the two activities of RecQL5 may be partially redundant. To investigate this possibility, we generated a double-point mutant that inactivates both the Pol II interaction and the helicase activity. Cells expressing this double mutant display no detectable correction of SCE and camptothecin phenotypes of RecQL5-/- DT40 cells, indicating these activities are partially redundant, and both are required for RecQL5 to protect genome stability in vivo. We found that RecQL5 also interacts with several other molecules important for DNA replication and repair. These include the RAD51 recombinase and PNCA (a clamp for DNA polymerase). We have mapped the interaction domains within RecQL5, and shown that mutants that are inactivated of these interactions have reduced capacity in suppressing SCE and resist campothecin-mediated cell killing. These studies suggest RecQL5 protects genome integrity through multiple mechanisms. We collaborated with other colleagues to investigate the in vivo association of the five human RecQ helicases with three well-characterized human replication origins. We show that only RECQ1 (also called RECQL or RECQL1) and RECQ4 (also called RECQL4) associate with replication origins in a cell cycle-regulated fashion in unperturbed cells. RECQ4 is recruited to origins at late G(1), after ORC and MCM complex assembly, while RECQ1 and additional RECQ4 are loaded at origins at the onset of S phase, when licensed origins begin firing. Both proteins are lost from origins after DNA replication initiation, indicating either disassembly or tracking with the newly formed replisome. Nascent-origin DNA synthesis and the frequency of origin firing are reduced after RECQ1 depletion and, to a greater extent, after RECQ4 depletion. Depletion of RECQ1, though not that of RECQ4, also suppresses replication fork rates in otherwise unperturbed cells. These results indicate that RECQ1 and RECQ4 are integral components of the human replication complex and play distinct roles in DNA replication initiation and replication fork progression in vivo. Single-strand DNA (ssDNA)-binding proteins (SSBs) are conserved from archaea to human, and play essential roles for a variety of DNA metabolic processes, including DNA replication, recombination, DNA damage detection and repair. Human SSB1 (hSSB1) has been shown to play an important role in the cellular DNA damage response. In bioinformatics searches of the human genome, we noticed that human genome contains a homolog of hSSB1, hSSB2. We hypothesized that hSSB1 and hSSB2 may form complexes similar to other OB-fold complexes, such as RPA. We immunoprecipitated complexes containing both hSSB1 and hSSB2. We found that these two proteins form two separate complexes. Interestingly, both complexes contain two identical components, named INTS3 and SSBIP1. We have performed siRNA depletion, and found that cells depleted of these proteins have reduced efficiency of homologous recombination-dependent repair of DSB, sensitivity to DNA damaging agents, and deficieny in ATM-dependent signaling. Our data demonstrate that these two new complex play important roles in protecting genome integrity in human. In a study of the origin of DNA damage, we participated in a collaborative project to study how mitochondria produce reactive oxygen that can modify DNA.
