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. However, the mechanisms that are responsible for GCR formation were not studied in depth. One of the major reasons for this is that many genes that suppress and enhance GCR formation have not yet been discovered. 1) Determine the role of RAD5 orthologs in mammalian GCR and further dissect the RAD5 pathway upstream signals and additional factors Persistent stalled replication forks collapse and cause genomic instability that can lead to cell death if unrepaired. In yeast, stalled replication forks are resolved either by bypassing DNA damage with translesion synthesis (TLS) polymerases or by TS to the nascent strand of the sister chromatid. Different modifications of Proliferating Cell Nuclear Antigen (PCNA) determine the bypass mechanisms. PCNA functions to load different DNA polymerases or DNA repair machinery on DNA. PCNA is monoubiquitinated by RAD18 for damage bypass by TLS, and further poly-ubiquitinated by RAD5 on the monoubiquitinated PCNA for currently uncharacterized TS pathways. We found that yeast Rad18 and Rad5 suppress GCR through the poly-ubiquitination of PCNA. Although the RAD18-dependent TLS pathway was studied extensively in yeast and mammals, the existence of RAD5 and PCNA polyubiquitination pathways in mammals has not been investigated, mainly because no mammalian RAD5 homolog has been identified. We hypothesized that the RAD5 pathway for TS existed in mammals and suppressed GCR. Although we could not find a RAD5 homolog using conventional sequence homology searches, with the help of the NHGRI Bioinformatics Core, we used the SMART search (http://smart.embl-heidelberg.de/) that finds orthologs based on domain structures. We found two genes, SHPRH and HLTF, as putative RAD5 orthologs. We confirmed that SHPRH is an ortholog of yeast RAD5 by demonstrating: 1) SHPRH promotes methyl-methane-sulfonate (MMS)-induced PCNA polyubiquitination at lysine 164; 2) SHPRH associates with human PCNA, RAD18, and the ubiquitin-conjugating enzyme UBC13; and 3) the inactivation of SHPRH by shRNA increases sensitivity to MMS and enhances chromosome breaks and abnormal chromosome structures in human cells. We next hypothesized that mice deficient in SHPRH would show a high incidence of tumorigenesis. Using a BayGenomics embryonic stem cell line, which contains a retroviral insertion at the mouse SHPRH locus, we have recently generated shprh-/-mice that we are currently monitoring for occurrence of tumorigenesis. 2) ELG1: determine whether alternative Replication Factor C (RRFC) complex protein directs DNA repair pathways and communicates with cell cycle checkpoints To investigate whether the role of ELG1 in GCR suppression is conserved in mammals, we cloned the human ELG1 gene by conducting a sequence homology search in the human genome database with help from the NHGRI Bioinformatics Core. When the expression of the human ELG1 gene was reduced by shRNA, an increase in 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 ELG1 expression at S-phase and after treatment of cells with various DNA-damaging agents, including MMS, hydroxyurea, aphidicolin, and gamma-irradiation. Finally, we found that the induction of ELG1 protein levels was caused by ATR-dependent inhibition of protein degradation. A manuscript describing these results is in preparation. Based on these observations, we decided to investigate 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 is lethal at an early developmental stage 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 (HR)-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 twelve proteins participating in a DNA damage response network with BRCA1 and BRCA2. FANCM is a recently identified component of the FA complex that is hypothesized to function at an early step of the FA pathway. MPH1 enhanced GCR formation when overexpressed, and the GCR rate was further elevated by additional mutations in the TLS, TS, and HR pathways. We demonstrated that the GCR enhancement by MPH1 overexpression was caused by the partial inactivation of HR. Analysis of MPH1 proteins carrying a different mutation in ATPase or helicase domains demonstrated that the GCR enhancement by MPH1 overexpression was not accomplished through ATPase or helicase activity but through its interaction with the RAD52-dependent HR pathway.