DNA damage from environmental sources as well as from the cellular metabolism itself threatens the integrity of the genome if not detected and removed. Moreover, many human genetic diseases that involve defects in DNA repair are associated not only with strongly elevated cancer predisposition but also with severe developmental, neurological and immunological abnormalities and/or symptoms of premature aging.
Studies in the Cooper lab have a dual emphasis: (1) to provide high resolution information on the molecular mechanisms of DNA repair processes that maintain the integrity of the genome, and (2) to investigate the possibility that the implied requirement for DNA repair processes for normal development, in addition to their role in cancer avoidance, is related to damage to DNA from reactive oxygen species generated during metabolism, and in particular to lesions that block transcription or replication. Because of qualitative similarity in the lesions induced by ionizing radiation (IR) and by cellular oxidation, understanding the mechanisms for repair of IR damage can contribute to understanding this essential role of DNA repair. There is thus expected to be substantial overlap between cellular responses to low dose IR and to endogenously generated oxidative damage.
DNA damage that blocks transcription poses a major threat to cellular viability, and a preferential repair process that rapidly removes lesions from transcribed strands of active genes is an important cellular defense mechanism. A major research focus of the Cooper lab is on elucidating the molecular mechanisms and cellular regulation of transcription-coupled repair (TCR) in mammalian cells and its connections to repair machinery from several different pathways. We use a multidisciplinary approach that integrates information from protein biochemistry and enzymology, molecular biology, cell biology, and structural biology.
Our emphasis is on characterization of the multiple critical functions of the highly pleiotropic human DNA repair protein XPG, since our evidence suggests that it plays a coordinating role in multiple repair processes for DNA damage, and on its partner proteins CSB and TFIIH. The enzymatic activity of XPG is strictly required in nucleotide excision repair (NER) for making the first incision in removal of UV and bulky carcinogen damage, and defects in this function result in the highly cancer-prone disease xeroderma pigmentosum (XP). XPG also plays important non-enzymatic roles both in base excision repair (BER) of oxidative DNA damage through coordination and stimulation of early steps in lesion removal and in TCR through interaction with stalled RNA polymerase, other TCR proteins including CSB and TFIIH, and transcription bubbles. Mutations in XPG that inactivate its non-enzymatic functions result in the severe postnatal developmental disorder Cockayne syndrome (CS). One or more of these non-enzymatic XPG functions is essential after birth, since mice and humans with knockout or severe truncations of XPG die in very early life, but the molecular basis for this requirement is not clear.
We are investigating the possibility that XPG is required for cellular responses to DNA lesions from endogenous sources that increase postnatally, e.g. damage from reactive oxygen species, and that unresolved intermediates in either BER or TCR of oxidative damage are particularly deleterious. Whether the role of XPG in BER is functionally connected to its role in early steps of TCR in this regard is a question of major interest that we are actively pursuing.
Our investigation of the multiple protein-protein interactions of XPG has tied the TCR pathway to other important processes for maintaining genomic integrity, including non-homologous end-joining, homologous recombination, structure-specific helicase action, and chromatin assembly. In addition, preliminary evidence suggests that BER of oxidative damage is inducible by low dose IR and requires TCR proteins including both XPG and CSB, and whether this inducible repair process underlies the adaptive response to IR is also of interest.
Querol-Audí J, Yan C, Xu X, Tsutakawa SE, Tsai MS, Tainer JA, Cooper PK, Nogales E, Ivanov I. Repair complexes of FEN1 endonuclease, DNA, and Rad9-Hus1-Rad1 are distinguished from their PCNA counterparts by functionally important stability. Proc Natl Acad Sci U S A. 2012 May 29;109(22):8528-33.
Trego KS, Chernikova SB, Davalos AR, Perry JJ, Finger LD, Ng C, Tsai MS, Yannone SM, Tainer JA, Campisi J, Cooper PK. The DNA repair endonuclease XPG interacts directly and functionally with the WRN helicase defective in Werner syndrome. Cell Cycle. 2011 Jun 15;10(12):1998-2007.
Tsutakawa SE, Classen S, Chapados BR, Arvai AS, Finger LD, Guenther G, Tomlinson CG, Thompson P, Sarker AH, Shen B, Cooper PK, Grasby JA, Tainer JA. Human flap endonuclease structures, DNA double-base flipping, and a unified understanding of the FEN1 superfamily. Cell. 2011 Apr 15;145(2):198-211.
Campeau E, Ruhl VE, Rodier F, Smith CL, Rahmberg BL, Fuss JO, Campisi J, Yaswen P, Cooper PK, Kaufman PD. A versatile viral system for expression and depletion of proteins in mammalian cells. PLoS One. 2009 Aug 6;4(8):e6529.
Fan L, Fuss JO, Cheng QJ, Arvai AS, Hammel M, Roberts VA, Cooper PK, Tainer JA. XPD helicase structures and activities: insights into the cancer and aging phenotypes from XPD mutations. Cell. 2008 May 30;133(5):789-800.
Pluth JM, Yamazaki V, Cooper BA, Rydberg BE, Kirchgessner CU, Cooper PK. DNA double-strand break and chromosomal rejoining defects with misrejoining in Nijmegen breakage syndrome cells. DNA Repair (Amst). 2008 Jan 1;7(1):108-18.
Tsutakawa SE, Hura GL, Frankel KA, Cooper PK, Tainer JA. Structural analysis of flexible proteins in solution by small angle X-ray scattering combined with crystallography. J Struct Biol. 2007 May;158(2):214-23.
Fuss JO, Cooper PK. DNA repair: dynamic defenders against cancer and aging. PLoS Biol. 2006 Jun;4(6):e203.
Perry JJ, Yannone SM, Holden LG, Hitomi C, Asaithamby A, Han S, Cooper PK, Chen DJ, Tainer JA. WRN exonuclease structure and molecular mechanism imply an editing role in DNA end processing. Nat Struct Mol Biol. 2006 May;13(5):414-22.
Fan L, Arvai AS, Cooper PK, Iwai S, Hanaoka F, Tainer JA. Conserved XPB core structure and motifs for DNA unwinding: implications for pathway selection of transcription or excision repair. Mol Cell. 2006 Apr 7;22(1):27-37.
Sarker AH, Tsutakawa SE, Kostek S, Ng C, Shin DS, Peris M, Campeau E, Tainer JA, Nogales E, Cooper PK. Recognition of RNA polymerase II and transcription bubbles by XPG, CSB, and TFIIH: insights for transcription-coupled repair and Cockayne Syndrome. Mol Cell. 2005 Oct 28;20(2):187-98.
Weinfeld M, Xing JZ, Lee J, Leadon SA, Cooper PK, Le XC. Factors influencing the removal of thymine glycol from DNA in gamma-irradiated human cells. Prog Nucleic Acid Res Mol Biol. 2001;68:139-49.
Fouladi B, Waldren CA, Rydberg B, Cooper PK. Comparison of repair of DNA double-strand breaks in identical sequences in primary human fibroblast and immortal hamster-human hybrid cells harboring a single copy of human chromosome 11. Radiat Res. 2000 Jun;153(6):795-804.
Löbrich M, Cooper PK, Rydberg B. Joining of correct and incorrect DNA ends at double-strand breaks produced by high-linear energy transfer radiation in human fibroblasts. Radiat Res. 1998 Dec;150(6):619-26.
Nouspikel T, Lalle P, Leadon SA, Cooper PK, Clarkson SG. A common mutational pattern in Cockayne syndrome patients from xeroderma pigmentosum group G: implications for a second XPG function. Proc Natl Acad Sci U S A. 1997 Apr 1;94(7):3116-21.
Löbrich M, Rydberg B, Cooper PK. Repair of x-ray-induced DNA double-strand breaks in specific Not I restriction fragments in human fibroblasts: joining of correct and incorrect ends. Proc Natl Acad Sci U S A. 1995 Dec 19;92(26):12050-4.