Transmitting genetic information without creating deleterious genetic alterations is one of the most important tasks. Cells have evolved systems that check for and repair potentially lethal DNA damage. However, when these systems do not work properly, DNA damage accumulates and causes genetic changes or cell death. Accumulation of genetic changes, which is defined as a genomic instability is frequently observed in various types of genetic disorders including cancers. Genomic instability has been documented as a preceding step for multiple inactivations of tumor suppressor genes and activations of proto-oncogenes. One type of genomic instability observed frequently in many cancers is gross chromosomal rearrangement (GCR). GCR includes translocations, deletions of chromosome arm, interstitial deletions, inversions, amplifications, chromosome end-to-end fusion and aneuploidy. Although little is known about the origin and mechanisms of GCRs observed in cancer cells, recent studies on genes mutated in inherited cancer predisposition syndromes have started to demonstrate that proteins that function in DNA damage responses, DNA repair, and DNA recombination, play crucial roles in the suppression of spontaneous and/or DNA damage-induced GCRs. The recent identification of strong correlations between genes responsible for genetic diseases including cancers and GCRs started to pinpoint the importance of GCRs. To understand mechanisms how GCRs are generated and how such GCR formation can lead tumorigenesis, we screened the entire yeast genome for mutations or overexpression that increase the rate of GCR formation. RAD5 and ELG1 from mutation screening and MPH1 from overexpression screening were selected for further studies of molecular mechanisms of these proteins to protect genome from deleterious GCR formation. 1. Determine the role of RAD5 orthologs in mammalian GCR and further dissect the RAD5 pathway upstream signals and additional factors. Previously, we identified two RAD5 orthologs in mammals and demonstrated that RAD5 orthologs, SHPRH and HLTF function to prevent collapse of persistent stalled replication forks by assisting template switching DNA damage bypass mechanism that uses the nascent strand of the sister chromatid for recombination mechanism for damage bypass. Among different modifications of Proliferating Cell Nuclear Antigen (PCNA) that determine the bypass mechanisms, we demonstrated that PCNA is poly-ubiquitinated by SHPRH and HLTF. Last year, we hypothesized that mice deficient in SHPRH would show a high incidence of tumorigenesis. We found that shprh-/- and hltf-/- and double knockout mice did not observe high level of tumorigenesis. In collaboration with Dr. Heinz Jacobs, we found that there is a redundant pathway that can complement the lack of SHPRH/HLTF pathway. SHPRH has a unique histone interaction domain called PHD domain. We recently found that this domain is important for HLTF as well as SHPRHs localization in the nucleolus. The localized SHPRH/HLTF in the nucleolus regulate the transcription of rRNA and assist cellular proliferation. We are currently investigating the link between DNA damage response of SHPRH/HLTF in their affects in rRNA transcription and proliferation. We also hypothesized that mice defective in SHPRH would lead problem in rRNA transcription related phenotype such as aging and autophagy. We are currently investigating these possibilities using mice model. 2. ELG1: determine whether alternative Replication Factor C (RFC) complex protein directs DNA repair pathways and replication. To investigate whether the role of ELG1 in GCR suppression in yeast is conserved in mammals, we cloned the human ELG1 gene (ATAD5) by conducting a sequence homology search in the human genome database. Previously, we demonstrated that the reduced expression of the ATAD5 gene by shRNA increased spontaneous DNA damage resulted as evidenced by an increase of phosphorylated histone H2Ax and ATM foci. The ELG1 protein was localized at the stalled replication fork after hydroxyurea treatment. We also demonstrated an increase of human ATAD5 expression at S-phase and after treatment of cells with various DNA-damaging agents, including MMS, hydroxyurea, aphidicolin, and gamma-irradiation. Previously, we found that ELG1 interacts with PCNA and USP1 that removes ubiquitin from PCNA after DNA damage bypass pathway. Last year, we investigated whether mice deficient in ELG1 would show a high incidence of tumorigenesis. In an attempt to create homozygous mice by using a retroviral insertion BayGenomics embryonic stem cell line, we found that the null mutation of mouse ELG1 was lethal at an early developmental stage. We recently confirmed such embryonic lethality could be slightly delayed in ATAD5 deficient zebrafish model. Most strikingly, we found that heterozygous atad5 mice developed high incidence of spontaneous tumors. In addition, in collaboration with Dr. Daphne Bells group in NHGRI, we found human somatic mutations of ELG1 gene in many endometrial tumors. We are currently testing whether these rare mutations found in endometrial tumors affect ATAD5s molecular function and cause phenotypes observed in mice and zebrafish. In addition to its role in DNA repair, we found that ATAD5 functions in DNA replication through its interaction with PCNA. We are currently investigating ATAD5s role in DNA replication, especially in lagging strand synthesis. 3. Determine the role of Mph1, the yeast homolog of FANCM, in DNA repair By screening genes that enhance GCR formation when overexpressed, we identified MPH1 as the strongest GCR enhancing gene. MPH1 has been implicated in a homologous recombination-dependent DNA repair pathway. Recently, the human homolog of MPH1 was discovered as the gene mutated in FA complementation group M (FANCM) patients. FA is a genomic instability disorder clinically characterized by congenital abnormalities, progressive bone marrow failure, and predisposition to malignancy. The FA core complex consists of fourteen proteins participating in a DNA damage response network with breast cancer susceptible proteins, BRCA1 and BRCA2. Based on homology between MPH1 and FANCM, we hypothesized yeast also has a FA like pathway that functions for repair of intercrosslink (ICL) repair. ICLs covalently link complementary DNA strands, block DNA replication and transcription, and must be removed to allow cell survival. We genetically characterized a conserved yeast ICL repair pathway comprised of the yeast homologs (Mph1, Chl1, Mhf1, Mhf2) of four FA proteins (FANCM, FANCJ, MHF1, MHF2). We found that this pathway is epistatic with Rad5-mediated DNA damage bypass and distinct from the ICL repair pathways mediated by Rad18 and Pso2 that remove ICL damage by different mechanisms. In addition, consistent with FANCMs role in stabilizing ICL-stalled replication forks, we found that Mph1 prevents ICL-stalled replication forks from collapsing into double strand breaks. This unique repair function of Mph1 is specific for ICL damage and does not extend to other types of damage. These studies reveal the functional conservation of the FA pathway and validate the yeast model for future studies to further elucidate the mechanism of the FA pathway.