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.
Fuss JO and Cooper PK. DNA repair: Dynamic defenders against cancer and aging. PLoS Biology 4, e203 (2006).
Sarker AH, Tsutakawa SE, Kostek S, Ng C, Shin DS, Peris M, Campeau E, Tainer JA, Nogales E, and 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 20, 187-198 (2005).
Fan L, Arvai A, 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 22, 27-37 (2006).
Perry JJP, Yannone SM, Holden LG, Hitomi C, Asaithamby A, Han S, Cooper PK, Chen DJ, and Tainer JA. WRN exonuclease structure and molecular mechanism imply an editing role in DNA end processing. Nature Struct. Mol. Biol. 13, 414-422 (2006).
Kalogeraki VS, Tornaletti S, Cooper PK, and Hanawalt PC. Comparative TFIIS-mediated transcript cleavage by mammalian RNA polymerase II arrested at a lesion in different transcription systems. DNA Repair 4, 1075-1087 (2005).
Wang J, Pluth JM, Cooper PK, Cowan MJ, Chen DJ, and Yannone SM. Artemis Deficiency Confers a DNA Double-Strand Break Repair Defect and Artemis Phosphorylation Status is Altered by DNA Damage and Cell Cycle Progression. DNA Repair 4, 556-570 (2005).Wang J-Y, Sarker AH, Cooper PK, and Volkert MR. The single-strand DNA binding activity of human PC4 prevents mutagenesis and killing by oxidative DNA damage. Mol. Cell. Biol. 24, 6084-6093 (2004).