A bright Quantum of Time in the Cleaver Laboratory; a Tribute and Retrospective

Many of my generation can remember the decline of the Carter administration and the ascendance of Reagan, a period of political history when many brilliant scientists roamed the halls of the Laboratory of Radiobiology and Environment Health working within or in the vicinity of Dr. James Cleaver’s laboratory. In this retrospective essay I will describe the people in this place at this time and how their work there produced intellectual ripples that continue to propel science today. This intellectual legacy serves as a tribute to Jim’s scientific influence. I joined Jim’s laboratory in the spring of 1979 and found a desk between Dr. William Bodell and Dieter Gruenert. My assigned laboratory bench was next to Dr. Leona Sampson. Down the hall were Drs. Jeffrey Schwartz and Sheila Galloway, who were working with Dr. Sheldon Wolff. Still further away up a flight of stairs was Dr. Robert Painter. Students of the DNA damage response will know all these names by their many seminal publications. I will review here several papers that they produced in this era that were connected to Dr. Cleaver. Hindsight suggests that my three and a half year tenure in the Cleaver laboratory represented a time of intense scholarly activity with discoveries that still attract our attention and focus current research. Bill Bodell came to the Cleaver laboratory from across the bay, having done his first postdoctoral fellowship with Dr. Beatrice Singer at UC-Berkeley. He was interested in how nucleosome structure might influence DNA alkylation and DNA repair. Several studies had indicated that DNA excision repair modified the structure of the nucleosome in the vicinity of the DNA lesion and Bill used micrococcal nuclease, exonuclease III and S1 nuclease to probe the dynamic changes in chromatin structure during repair of UV-induced DNA damage. The product of DNA repair replication immediately after UV treatment was in a nuclease-accessible state, suggesting there was an alteration of nucleosome structure at the site of repair (1). With time after synthesis the repair patch became nuclease-resistant implying the re-establishment of a nucleosome at the site. Further analysis of the repair patch synthesized in the presence of cytosine arabinoside (araC) indicated that the cytosine analog was not a complete chain terminator and some repair patches were ligated with incorporated araC. These araC-labeled repair patches were resistant to digestion with exonuclease III, which is a 30 exonuclease. Our data showed that nucleosome structure was altered at sites of nucleotide excision repair (NER) but could not distinguish between complete or partial removal of nucleosome structure at the site of damage. The discovery of chromatin remodeling complexes that can slide nucleosomes along DNA supports a model in which the nucleosome is moved away from the damage site to allow access by the core NER factors. The demonstration that damaged DNA binding protein (DDB) made up of DDB1 and XPE/DDB2 is required for repair of UV-induced damage in heterochromatin suggests that DDB might participate in recognition of damaged sites in chromatin (2,3). The demonstration that the SWI/SNF chromatin remodeling complex also enhances NER in chromatin supports a model with DDB and SWI/SNF serving to slide nucleosomes away from damaged DNA to allow binding by the core components of NER. After completion of repair SWI/SNF may slide the nucleosome back to its original position. Equally plausible is a model with disassembly of nucleosomes. A recent paper from Goldstein et al. (4) demonstrates that nucleolin can enforce the removal of H2A and H2B at the site of a DNA double strand break (DSB), with H3 and H4 being retained at the break. Replacement of H2A and H2B at the site of the DSB occurred with kinetics similar to the rate of rejoining of the DSB. Thus, nucleosome sliding and disassembly/reassembly have been demonstrated to facilitate DNA repair in chromatin. It is up to the next generation of DNA repair scientists to determine the steps of nucleosome remodeling during NER. Dieter Gruenert was a graduate student at UC-Berkeley but studying under Jim’s mentorship. He was interested in the role of xeroderma pigmentosum (XP) and Fanconi anemia (FA) factors in the repair of psoralen-induced DNA interstrand crosslinks (5,6). For his dissertation research Dieter measured the formation and repair of DNA monoadducts produced by reaction of angelicin (a monofunctional form of psoralen) with DNA in the presence of near UV light and DNA interstrand crosslinks produced by reaction of the bifunctional 8-methoxypsoralen with DNA in the presence of near UV light. A normal human fibroblast line and a FA fibroblast line were found to repair the psoralen monoadducts produced by angelicin while a complementation group A XP fibroblast line could not. In contrast, both the FA and XP fibroblasts were defective in repair of psoralen DNA interstrand crosslinks. This work confirmed that FA cells were defective in repair of DNA interstrand crosslinks, but expressed normal NER for monoadducts, and demonstrated that an NER factor missing in group A XP cells was also required for repair of DNA interstrand crosslinks. The mechanisms of crosslink repair have attracted considerable attention over the last 30 years with studies showing that NER factors interact with FA gene products particularly in S phase cells to rid the genome of interstrand crosslinks (7). The evolutionary basis for this complex biochemistry has recently become apparent with the demonstration that endogenous metabolites acetaldehyde and formaldehyde can react with nuclear DNA to produce interstrand crosslinks (8,9). Remarkably, repair of such lesions may be critical for protection against disabilities such as cancer, fetal alcohol syndrome and hematopoietic failure. †This paper is part of the Special Issue honoring James Cleaver. DOI: 10.1111/php.12384 © 2015 The American Society of Photobiology