Biochemist who invented recombinant DNA technology. Biochemist who invented recombinant DNA technology.

Nucleotide excision repair (NER) is a fundamental DNA repair process that is ubiquitously distributed from bacteria to humans. The process is essential for the enzyme-catalyzed removal of various types of base damage from the nuclear genome, typically those that result in significant distortion of the helical structure of DNA. This article discusses the overview and biology of NER.

Mischievous steward of molecular biology’s golden age. Mischievous steward of molecular biology’s golden age.

This article reviews the early history of the discovery of an DNA repair pathway designated as base excision repair (BER), since in contrast to the enzyme-catalyzed removal of damaged bases from DNA as nucleotides [called nucleotide excision repair (NER)], BER involves the removal of damaged or inappropriate bases, such as the presence of uracil instead of thymine, from DNA as free bases.

The past year or so has witnessed the templates, with emergence of a plethora of prokary10-4 (19), substa otic and eukaryotic genes that are known of most replicati or predicted to encode previously unidenvitro (13). tified DNA polymerases (1-13). Some of In addition to these are members of an extended superpolymerase calle family of prokaryotic and eukaryotic proE. coli, the prod teins called the UmuC/DinB nucleotidyl a member of tl transferase superfamily, named after early purified recently discovered prokaryotic members (14, 15). IV is devoid of This superfamily is represented presently tivity and is stric by the UmuC, DinB, Rad30, and Revl Significantly, wi subfamilies (14, 15). More recently idenspontaneous mu tified polymerases resemble DNA replizyme introduce cative enzymes, and others seem to be aligned primerrelated to the Pol P and terminal transsulting in -1 fr; ferase proteins. Most recently, il A current working hypothesis is that purified maltosi when highly processive semiconservative fusion protein is DNA replication is arrested at lesions in thymine-thymin DNA, the replicative machinery is disproducts, or abas placed from the replication fork and rein vitro (19). Cl placed by these DNA polymerases. When merase V, E. col the offending lesion has been bypassed not able to suppl successfully, the polymerase displaceically altered ba ment/replacement process is reversed, support DNA syi and the replication machinery continues lication forks. high-fidelity, highly processive DNA synThe spotlight thesis. There is substantial evidence that functions of som

This article reviews the early history of the discovery of an DNA repair pathway designated as base excision repair (BER), since in contrast to the enzyme-catalyzed removal of damaged bases from DNA as nucleotides [called nucleotide excision repair (NER)], BER involves the removal of damaged or inappropriate bases, such as the presence of uracil instead of thymine, from DNA as free bases.

There is no shortage of written contributions on various aspects of the history of genetics and molecular biology—The Eighth Day of Creation: The Makers of the Revolution in Biology stands out as a premier example, memorably focusing on reaching the intelligent lay reader. Matthew Cobb’s contribution is unquestionably written for scientists. But it too deserves adulation as a masterwork. The introductory chapter Genes before DNA reminds readers of the familiar early pioneers of genetics, including Gregor Mendel, Hugo de Vries, Theodor Boveri, Wilhelm Johannsen, Thomas Hunt Morgan, and Nikolai Koltsov. This chapter also thoughtfully informs us of the important intellectual contributions of the eminent physicist Erwin Schrodinger, who is credited with the first notion of a “code script” when talking about how genes operate. The succeeding chapter called Information Is Everywhere may tempt all but the most intellectually oriented readers to toss the book aside. My advice is to curb this impulse should it arise! This reviewer was particularly entranced by Cobb’s treatment of the famous transformation experiments executed by Oswald Avery and his colleagues Colin MacLeod and Macleod McCarthy in the mid-1940s that led them to the conclusion that genetic information resides in DNA rather than proteins, the latter being the alternative and widely held view in the genetics community. The erudition of this chapter lies in an element that particularly distinguishes Cobb’s writing, namely the historical depth that he has brought to this literary contribution. Most, if not all, students are taught that Avery was the first to experimentally demonstrate that genes are made of DNA. But few are likely aware of the enormous challenges that he had to endure from the unshakable adherence to the entrenched notion that genes are made of protein and that, if DNA was in anyway involved in gene action, it was surely by way of some subsidiary (perhaps structural) role. Besides, DNA was then considered an utterly boring molecule equipped with just the four bases, deoxyribose and phosphate, hardly persuasive “to bring about the almost infinitely different effects produced by genes.” Cobb informs that both the experiments of Avery and his group and the equally famous later experiments of Alfred Hershey and Martha Chase were persistently dogged by a criticism that was essentially impossible to definitively address, namely that they could never definitively prove that their transforming principle contained absolutely no protein. Cobb reveals that the influential biologist Alfred Mirsky, together with Arthur Pollister, published a widely read article that stressed that “there can be little doubt in the mind of anyone who has prepared nucleic acids that traces of protein probably remain in even the best preparations and that as much as 1 or 2 per cent of protein could be present in a preparation of pure, protein-free nucleic acid.” Even the celebrated geneticist Herman Muller wrote in an article that he was personally convinced that Mirsky’s suggestion that undetected “genetic proteins floating free in the medium caused Avery’s results.” Cobb points out that, regardless of “the overwhelming evidence, all of which suggested that the transforming principle was made of DNA and that genes may be too,” the final paragraph of the paper in which Avery and his colleagues announced their startling findings “opened with a phrase that suggested that the team was not quite as confident as they ought to have been.” “It is of course possible that the biological activity of the substance described here is not an inherent property of the nucleic acid but is due to minute amounts of some other substance adsorbed to it or so intimately associated with it as to escape detection,” Avery et al. wrote. But, they also boldly stated, “there is no evidence in favor of such a hypothesis that is chiefly supported by the traditional view that nucleic acids are devoid of biological specificity.” Distressingly, this traditional view hung around in the minds of many scientists, even prominent ones, for years, causing Avery to suffer frank clinical depression. And when Avery died in 1955, “the brief obituary that appeared in the New York Times did not even mention DNA.” Regardless, over the years, Avery’s contention stimulated the thinking and work of an increasing cadre of established and future stars in genetics, including Joshua Lederberg. Cobb notes that the journal Nature described Avery’s work in glowing terms, and a (small) number of scientists were in fact highly complementary. In October, 1944, the New York Academy of Medicine awarded Avery its Gold Medal. And in 1945, the Royal Society of London graced his experimental achievements with the Copley Medal. But Avery was never graced with the highly deserved distinction of Nobel Laureate. Cobb interrupts the progress of his history with another epistemological chapter dubbed The Age of Control in which he outlines the discipline of cybernetics, a term that it is relevant to the study of systems, including mechanical, physical, biological, cognitive, and social systems. Cybernetics is applicable when a system being analyzed incorporates a closed signaling loop—i.e., where action by the system generates some change in its environment and that change is reflected in the system in some manner (feedback) that triggers a system change. The intent of this chapter is to alert the reader to the emergence of cybernetics when feedback mechanisms in molecular biology were discovered by later makers and shakers in molecular biology, notably from the exquisite experiments on gene regulation executed by the famous French duo of Francois Jacob and Jacques Monod described in a later chapter. Much of the rest of the book covers the history on the elucidation of the structure of DNA (a topic well covered in James D. Watson’s The Double Helix) and the pursuit of the Holy Grail—deciphering the genetic code. Cobb peppers his writing of the latter seminal breakthrough with delightfully interesting anecdotal information that displays the depth of research for his book. He informs the reader: “On March 19, 1953, about two weeks after the double helix model had been completed, Francis Crick wrote a letter to his 12-year old son, Michael, who was at boarding school. Crick told Michael what he had discovered, and included a sketch of the structure of DNA. He then went on to explain the significance of the double helix. ‘It’s like a code,’ Crick wrote to Michael. ‘If you are given one set of letters you can write down the others. Now we believe that the D.N.A. is a code. That is, the order of the bases (the letters) makes one gene different from another gene (just as one page of print is different from another).’” Cobb relates that, while the notion that the sequence of bases in a DNA chain had been speculated for some time, this letter to his very young son was the first time that anyone had stated in writing that DNA contains a code. In 2013, the letter fetched $6 million at an auction! Crick’s leadership, intellectual genius, and scintillating personality during the period in which the genetic code was slowly but surely unraveled leap majestically from Cobb’s pen. Equally arresting and presumably little-known historical anecdotes surface when Cobb relates that, after Marshall Nirenberg (an unknown scientist to most of the molecular biology community) reported his use of homopolymers to elucidate the genetic code in a 10 min talk at the Fifth International Congress of Biochemistry in Moscow in August 1961, Matt Meselson informed Crick of these electrifying experimental results, prompting Crick to invite him to present his findings again in a longer plenary talk at a symposium that Crick was to chair the following day. Following his second presentation, Nirenberg was so gratified and elated he was prompted to comment: “The reception was really remarkable, fantastic. I remember Matt Meselson, who was sitting right up front. I didn’t know him at the time, but he was so overjoyed about hearing this stuff that he impulsively jumped up, grabbed my hand, and actually hugged me and congratulated me for doing that. I could have been part of a rock band or something. That really meant an awful lot to me. It really meant more to me than all kinds of awards and what-not because it was genuine and spontaneous.” The work in the Nirenberg laboratory and that of a competing laboratory led by the Spanish-born biochemist Severo Ochoa contributed mightily to deciphering the genetic code. In 1968, Nirenberg shared the Nobel prize in physiology or medicine with Robert Holley and Gobind Khorana. Cobb concludes his book with a section entitled Update that details the history of molecular genetics and molecular biology to the present time, including the discovery of introns, the use of the polymerase chain reaction (PCR), sequencing entire genomes, paleogenomics, population genetics, evolutionary genetics, genetic engineering, the potential for synthetic biology, and more. The book also features a pleasing gallery of photos. The dominance of individual brilliance in molecular genetics so remarkably displayed by Francis Crick and Sydney Brenner during the decades of 1950s and 1960s is fading all too rapidly. In his conclusion, Cobb addresses the perils of “big science,” especially in the field of genomics, pointing to a paper in Nature Genetics published in 2014 that listed 440 authors! Cobb notes “It is now becoming commonplace, changing the relationship of individual scientists to the work they produce, rendering each person’s contribution relatively minor and highly specific.” This threatening shift in the sociology of science cries out for attention if molecular biology is to regain its former attraction to college students interested in pursuing careers in disciplines exemplified by modern day genomics. All in all, Matthew Cobb, who hails from the University of Manchester—which notably includes a Center for the History of Science, Technology and Medicine and whose eclectic historical contributions include the efforts of the French resistance during WWII—has presented the scientific and perhaps members of the non-scientific communities an erudite and comprehensive history that should be required reading for all graduate students in the disciplines of genetics and molecular biology and, most certainly, students of the history of science.

REV1 is a eukaryotic member of the Y-family of DNA polymerases involved in translesion DNA synthesis and genome mutagenesis. Recently, REV1 is also found to function in homologous recombination. However, it remains unclear how REV1 is recruited to the sites where homologous recombination is processed. Here, we report that loss of mammalian REV1 results in a specific defect in replication-associated gene conversion. We found that REV1 is targeted to laser-induced DNA damage stripes in a manner dependent on its ubiquitin-binding motifs, on RAD18, and on monoubiquitinated FANCD2 (FANCD2-mUb) that associates with REV1. Expression of a FANCD2-Ub chimeric protein in RAD18-depleted cells enhances REV1 assembly at laser-damaged sites, suggesting that FANCD2-mUb functions downstream of RAD18 to recruit REV1 to DNA breaks. Consistent with this suggestion we found that REV1 and FANCD2 are epistatic with respect to sensitivity to the double-strand break-inducer camptothecin. REV1 enrichment at DNA damage stripes also partially depends on BRCA1 and BRCA2, components of the FANCD2/BRCA supercomplex. Intriguingly, analogous to FANCD2-mUb and BRCA1/BRCA2, REV1 plays an unexpected role in protecting nascent replication tracts from degradation by stabilizing RAD51 filaments. Collectively these data suggest that REV1 plays multiple roles at stalled replication forks in response to replication stress.

Photoreactivation, an enzyme-catalyzed reaction during which two covalently linked pyrimidine dimers in DNA are monomerized and restored to their native conformation was the first DNA repair mechanism to be discovered, an event that transpired in the late 1940's through the efforts of the American biologist Albert Kelner while at the Cold Spring Harbor Laboratories in upstate New York. The phenomenon was Independently observed by Renato Dulbecco shortly thereafter, then a post-doctoral fellow in Salvador Luria's Laboratory in Bloomington Indiana. However, Luria and Dulbecco yielded priority to Kelner's discovery.

The recognition that organisms mount physiological responses to DNA damage initially came from work on Escherichia coli and was surprising to many scientists. This chapter first traces the intellectual development of the present model for SOS regulation in prokaryotes and the identification of genes under SOS control. It considers various molecular mechanisms that are used to fine-tune the expression of individual SOS genes and summarizes our present understanding of the physiology of the SOS responses. The genetic studies of recA, lexA, recA mutants, and lexA mutants indicate the existence of the SOS system. Then, it presents essential elements of SOS transcriptional regulation. It also presents identifying SOS genes by the use of fusions, searching for potential lexA-binding sites and expression microarray analysis. A variety of experiments now support the unifying view that the ultimate signal for SOS induction in vivo is the generation of regions of ssDNA within the cell, which in turn results in the formation of sufficient RecA nucleoprotein filaments to mediate LexA cleavage. Next, the chapter briefly discusses additional subtleties in transcriptional regulation of the SOS responses, and known and putative SOS responses of E. coli from a physiological perspective. Other covered topics are SOS responses in pathogenesis, toxicology, and other bacteria.

This chapter discusses the molecular mechanism of nucleotide excision repair (NER) in eukaryotes, with emphasis on the reaction mechanism in mammalian cells and in the budding yeast, Saccharomyces cerevisiae. The NER mechanism is highly conserved in eukaryotes, and most components and features of the reaction mechanism are very similar in these two organisms. A cell-free system that reflects NER in mammalian cells was developed in the late 1980s and was followed by experimental approaches that measure repair synthesis or the excision of damage-containing oligonucleotide fragments in extracts from mammalian cells and yeast. These techniques have served to identify and track proteins required for NER and have provided specific assays for the purification of NER proteins. The results of genetic studies and the biochemical systems have facilitated the reconstitution of the mammalian and yeast NER machinery with purified protein components and DNA molecules containing single lesions placed at specific sites. To set the stage for the discussion to follow, it is useful to first summarize the reconstitution results. The incision step of NER in the yeast S.cerevisiae has been reconstituted with UV-irradiated DNA and a set of proteins comprising Rad14, Rad4-Rad23, RPA, TFIIH, and the nucleases Rad2 and Rad1-Rad10. The chapter also discusses mechanism of assembly and action of the NER machinery, modulation and regulation of NER in eukaryotes, and evolution of the eukaryotic NER system.

This chapter focuses solely on the human as an experimental organism. The data discussed are derived from unique experimental approaches. There is substantial evidence that cancer segregates in many additional families, albeit at a reduced frequency compared with that for the more severe syndromes. There is also epidemiological evidence for significant variation in DNA repair capacity among individuals in the population and evidence that those with mildly reduced capacity may be more likely to exhibit a cancer predisposition. A great many publications have proposed associations between specific genetic variants (polymorphisms) in DNA repair and/or damage response genes and a cancer predisposition. Evidence documenting the impact of a polymorphism on protein function is generally lacking. Thus, the appreciation of a specific role for the variant proteins in disease, while logical in theory, remains an important aspect of DNA repair and mutagenesis that is still under development.