Appropriate implementation of Okazaki fragment maturation during DNA replication in eukaryotic cells is a fundamental mechanism for avoidance of mutations and genome stability. During lagging strand DNA synthesis, multiple RNA primers and immediately adjoined DNA-fragments are synthesized by primase (a hetero tetramer of a RNA polymerase and DNA Pol ). However, both of the enzymes lack a proof reading function, different from the other DNA polymerases. Therefore, this initial RNA-DNA fragment (alpha- segment of the Okazaki fragment) is highly mutagenic and has to be processed by nuclease complexes. This proposal aims to define detailed molecular mechanism for the nuclease-driven RNA primer processing in eukaryotic nuclei and mitochondria. For the last funding period, we have defined the roles of several nucleases in the processes, including S. cerevisiae RNase H(35), ScRad27 or human FEN1, and exonuclease-1, and mutagenic consequences when these nucleases are defective. We have also accumulated solid evidence to demonstrate that nuclease helicase DNA2 exclusively localizes into mitochondria, and plays a vital role in RNA primer removal during mitochondrial DNA replication. These novel exciting observations prompted us to develop new and additional specific aims in this renewal application. The current proposal focuses to test a central hypothesis that 1-segment processing is a vital part of cellular mechanisms to maintain genomic integrity and prevent mutagenic stresses due to intrinsic DNA sequence obstacles and exogenous insults. Deficiency of this integrative machinery could lead to a high incidence of mutagenesis and carcinogenesis. We will further define detailed molecular mechanisms for the nuclease-driven "1-segment" processing in Okazaki fragment maturation in yeast and mammalian cell systems, during replication of normal DNA sequence and repetitive DNA sequence regions, in the nucleus as well as the mitochondrion. Through a series of vigorous systematic analyses, we intend to obtain a high resolution image of how these nuclease complexes collectively work towards RNA primer processing in different scenarios and to relate in vitro and in vivo data using yeast and mammalian systems, including human cell lines and transgenic mice. Information made available from this systematic study will establish a relationship between this mechanism, unique mutagenic phenotype(s), and development of cancers.
The current application aims to test whether 1-segment processing is a vital part of the cellular mechanism for maintaining genomic integrity and for preventing mutagenic stresses and cancers. The proposed studies should lead to advances in our understanding of early events and molecular mechanism of many cancer predispositions. The information made available via these studies will be useful in developing new strategies for cancer prevention and treatment.
|Ronchi, Dario; Di Fonzo, Alessio; Lin, Weiqiang et al. (2013) Mutations in DNA2 link progressive myopathy to mitochondrial DNA instability. Am J Hum Genet 92:293-300|
|Lin, Weiqiang; Sampathi, Shilpa; Dai, Huifang et al. (2013) Mammalian DNA2 helicase/nuclease cleaves G-quadruplex DNA and is required for telomere integrity. EMBO J 32:1425-39|
|Zheng, Li; Dai, Huifang; Hegde, Muralidhar L et al. (2011) Fen1 mutations that specifically disrupt its interaction with PCNA cause aneuploidy-associated cancer. Cell Res 21:1052-67|
|Zheng, Li; Shen, Binghui (2011) Okazaki fragment maturation: nucleases take centre stage. J Mol Cell Biol 3:23-30|
|Sun, Daxin; Zhou, Mian; Kowolik, Claudia M et al. (2011) Differential expression patterns of capping protein, protein phosphatase 1, and casein kinase 1 may serve as diagnostic markers for malignant melanoma. Melanoma Res 21:335-43|
|Zheng, Li; Jia, Jia; Finger, L David et al. (2011) Functional regulation of FEN1 nuclease and its link to cancer. Nucleic Acids Res 39:781-94|
|Guo, Zhigang; Zheng, Li; Xu, Hong et al. (2010) Methylation of FEN1 suppresses nearby phosphorylation and facilitates PCNA binding. Nat Chem Biol 6:766-73|
|Guo, Zhigang; Zheng, Li; Dai, Huifang et al. (2009) Human DNA polymerase beta polymorphism, Arg137Gln, impairs its polymerase activity and interaction with PCNA and the cellular base excision repair capacity. Nucleic Acids Res 37:3431-41|
|Guo, Zhigang; Chavez, Valerie; Singh, Purnima et al. (2008) Comprehensive mapping of the C-terminus of flap endonuclease-1 reveals distinct interaction sites for five proteins that represent different DNA replication and repair pathways. J Mol Biol 377:679-90|
|Zheng, Li; Zhou, Mian; Guo, Zhigang et al. (2008) Human DNA2 is a mitochondrial nuclease/helicase for efficient processing of DNA replication and repair intermediates. Mol Cell 32:325-36|
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