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. 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 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, SHPRH. 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. Although we tested for association of SHPRH and HLTF with tumorigenesis in vivo, the inactivation of these genes did not result in an increase in tumorigenesis. SHPRH has a unique histone interaction domain called PHD domain. We recently found that this domain is important for SHPRH localization in the nucleolus. In our characterization of the general DNA damage response regulating PCNA ubiquitination, we unexpectedly found that phosphorylation of Serine 4 and 8 of RPA32, which we used as a DNA damage response control, depends on DNA dependent protein kinase. We found a novel function of SHPRH in the nucleolus that drove the project into a novel molecular mechanism of SHPRH in rRNA transcription in the nucleolus. We found the function of SHPRH for rRNA transcription is mTOR-dependent. 2. ATAD5 (mammalian ELG1 homolog): determine whether alternative Replication Factor C (RFC) complex protein directs DNA repair pathways and replication. We reported that mice haploinsufficient in Atad5 showed a high incidence of tumorigenesis. We recently confirmed that embryonic day 7.5 to 8.5 as embryonic lethality caused by homozygous null mutation of ATAD5. In addition, in collaboration with Dr. Daphne Bells group in NHGRI, we found human somatic mutations of ATAD5 gene in many endometrial tumors. We also found several rare polymorphisms as well as cancer mutations in other tumor types. We recovered a zebrafish atad5 null allele from the TILLING project in the NHGRI zebrafish core facility. Unlike the atad5 null mouse, which dies at embryonic day 8.5, an atad5 null zebrafish survives until seven days post fertilization (dpf). The lethal phenotype of the atad5 null zebrafish became obvious right after hatching when fertilized eggs were treated with MMS. Therefore, we believe that in vivo functional activity of ATAD5 variants identified can be tested with the atad5 null zebrafish model. We have generated various ectopic expression constructs with a single sequence variant found in somatic cancer mutations as well as database search in the International Cancer Genome Consortium for the Cancer Genome Atlas using site-directed mutagenesis. We are currently testing whether these rare mutations found in affect ATAD5s molecular function and cause phenotypes observed in mice and zebrafish as well as in tissue culture system.

Project Start
Project End
Budget Start
Budget End
Support Year
12
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Human Genome Research
Department
Type
DUNS #
City
State
Country
Zip Code
Zanotti, Kimberly J; Maul, Robert W; Castiblanco, Diana P et al. (2015) ATAD5 deficiency decreases B cell division and Igh recombination. J Immunol 194:35-42
Kubota, Takashi; Myung, Kyungjae; Donaldson, Anne D (2013) Is PCNA unloading the central function of the Elg1/ATAD5 replication factor C-like complex? Cell Cycle 12:2570-9
Lee, Kyoo-young; Fu, Haiqing; Aladjem, Mirit I et al. (2013) ATAD5 regulates the lifespan of DNA replication factories by modulating PCNA level on the chromatin. J Cell Biol 200:31-44
Nguyen, Hai Dang; Becker, Jordan; Thu, Yee Mon et al. (2013) Unligated Okazaki Fragments Induce PCNA Ubiquitination and a Requirement for Rad59-Dependent Replication Fork Progression. PLoS One 8:e66379
Fox, Jennifer T; Sakamuru, Srilatha; Huang, Ruili et al. (2012) High-throughput genotoxicity assay identifies antioxidants as inducers of DNA damage response and cell death. Proc Natl Acad Sci U S A 109:5423-8
Daee, Danielle L; Ferrari, Elisa; Longerich, Simonne et al. (2012) Rad5-dependent DNA repair functions of the Saccharomyces cerevisiae FANCM protein homolog Mph1. J Biol Chem 287:26563-75
Fox, Jennifer T; Myung, Kyungjae (2012) Cell-based high-throughput screens for the discovery of chemotherapeutic agents. Oncotarget 3:581-5
Daee, Danielle L; Myung, Kyungjae (2012) Fanconi-like crosslink repair in yeast. Genome Integr 3:7
Park, H D; Lee, Y; Oh, Y K et al. (2011) Pancreatic adenocarcinoma upregulated factor promotes metastasis by regulating TLR/CXCR4 activation. Oncogene 30:201-11
Calvo, Katherine R; Vinh, Donald C; Maric, Irina et al. (2011) Myelodysplasia in autosomal dominant and sporadic monocytopenia immunodeficiency syndrome: diagnostic features and clinical implications. Haematologica 96:1221-5

Showing the most recent 10 out of 25 publications