The human genome is littered with sequences derived from transposable elements from the Hsmar1 transposon, but there is only one intact copy of the Hsmar1 transposase gene termed Metnase (also known as SETMAR) that exists within a chimeric SET-transposase fusion protein. Although Metnase retains most of the transposase activities, it has evolved as a double-strand break (DSB) repair protein in anthropoid primates. Metnase is localized on chromosome 3p26, a region of frequent abnormalities in various cancers and is highly expressed in most tissues and cell lines. Mutations in Metnase that cause early termination were found in many transformed cell lines, although clinical relevance of these mutations has not been established. Our long-term goal is to understand how a protein with transposase activity in humans promotes DSB repair and chromosome decatenation, and what role the SET domain may play. Given that Metnase requires both the SET and transposase domains for its function(s) in DSB repair, we hypothesize that the acquisition of new functions may have resulted from a chimeric fusion between transposase and the SET domains. In this study we proposed three specific aims to elucidate the mechanism of this human SET- transposase protein in DSB repair and chromosome decatenation.
Aim 1 : Determine the mechanism by which Metnase is localized to DSB sites. We will identify the region within the SET domain of Metnase crucial for localization to DSB sites following IR treatment. We will also investigate whether the interaction of Metnase with Pso4, a Metnase binding partner that plays a crucial role in Metnase localization at DSB sites, is crucial for Metnase localization at DSB sites. Finally, we will examine whether Metnase directly interacts with the Ku70/80 complex. If so, a mutant Metnase defective in interaction with Ku complex will be generated, and we will determine how this mutant differs from wt-Metnase in their localization at DSB sites.
Aim 2 : Determine the role(s) of Metnase's biochemical activities in DNA end joining and chromosome decatenation. Metnase not only possesses a structure-specific endonuclease and HLMT activities, but also interacts with Lig4, Pso4, and Topo II1, all of which could play role(s) in NHEJ repair and/or chromosome decatenation. We will examine how Metnse's biochemical activities are involved in DNA end joining and chromosome decatenation. First, we will substitute key amino acids within the catalytic site identified from the crystal structure of Metnase transposase domain and examine the mutants for DNA cleavage, DNA end processing, and end joining activities. Secondly, we will examine how Metnase's interaction with Lig4 affects recruitment of Lig4-XRCC4 to DSB sites and DNA end joining. Thirdly, we will examine how Metnase binding partner (Pso4) and its interaction with Metnase influence DNA end joining. Fourth, we will examine whether Metnase mutant(s) lacking HLMT and/or auto-methylation activity support stimulation of DSB repair. Finally, Metnase mutants lacking its biochemical activities will be examined for promotion of chromosome decatenation activity.

Public Health Relevance

We have isolated a novel protein termed Metnase that helps repair of radiation-induced chromosomes breaks and chromosome decatenation in humans. Understanding the molecular mechanism by which Metnase and its binding partners contribute to repair of chromosomal breaks and decatenation could not only help understand stable maintenance of human genome but also be useful in developing a drug that inhibits chromosomal DNA repair and decatenation in cancer cells.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA151367-02
Application #
8272533
Study Section
Cancer Etiology Study Section (CE)
Program Officer
Pelroy, Richard
Project Start
2011-06-03
Project End
2016-03-31
Budget Start
2012-04-01
Budget End
2013-03-31
Support Year
2
Fiscal Year
2012
Total Cost
$319,550
Indirect Cost
$112,050
Name
Indiana University-Purdue University at Indianapolis
Department
Biochemistry
Type
Schools of Medicine
DUNS #
603007902
City
Indianapolis
State
IN
Country
United States
Zip Code
46202
Kim, Hyun-Suk; Nickoloff, Jac A; Wu, Yuehan et al. (2017) Endonuclease EEPD1 Is a Gatekeeper for Repair of Stressed Replication Forks. J Biol Chem 292:2795-2804
Nickoloff, Jac A; Jones, Dennie; Lee, Suk-Hee et al. (2017) Drugging the Cancers Addicted to DNA Repair. J Natl Cancer Inst 109:
Kim, Hyun-Suk; Williamson, Elizabeth A; Nickoloff, Jac A et al. (2017) Metnase Mediates Loading of Exonuclease 1 onto Single Strand Overhang DNA for End Resection at Stalled Replication Forks. J Biol Chem 292:1414-1425
Chun, Changzoon; Wu, Yuehan; Lee, Suk-Hee et al. (2016) The homologous recombination component EEPD1 is required for genome stability in response to developmental stress of vertebrate embryogenesis. Cell Cycle 15:957-62
Hromas, Robert; Williamson, Elizabeth; Lee, Suk-Hee et al. (2016) PREVENTING THE CHROMOSOMAL TRANSLOCATIONS THAT CAUSE CANCER. Trans Am Clin Climatol Assoc 127:176-195
Chen, Qiujia; Georgiadis, Millie (2016) Crystallization of and selenomethionine phasing strategy for a SETMAR-DNA complex. Acta Crystallogr F Struct Biol Commun 72:713-9
Wu, Yuehan; Lee, Suk-Hee; Williamson, Elizabeth A et al. (2015) EEPD1 Rescues Stressed Replication Forks and Maintains Genome Stability by Promoting End Resection and Homologous Recombination Repair. PLoS Genet 11:e1005675
Kim, Hyun-Suk; Guo, Chunlu; Thompson, Eric L et al. (2015) APE1, the DNA base excision repair protein, regulates the removal of platinum adducts in sensory neuronal cultures by NER. Mutat Res 779:96-104
Kim, Hyun-Suk; Kim, Sung-Kyung; Hromas, Robert et al. (2015) The SET Domain Is Essential for Metnase Functions in Replication Restart and the 5' End of SS-Overhang Cleavage. PLoS One 10:e0139418
Kim, Hyun-Suk; Chen, Qiujia; Kim, Sung-Kyung et al. (2014) The DDN catalytic motif is required for Metnase functions in non-homologous end joining (NHEJ) repair and replication restart. J Biol Chem 289:10930-8

Showing the most recent 10 out of 17 publications