Replicating And Repairing The Genome: From Basic Mechanisms To Modern Genetic Technologies
and Beverley Fermor for valuable suggestions that helped make the book more accessible to newcomers to the field. Finally, I thank the Duke University Biochemistry Department for support and for providing an outstanding environment in which to pursue my research interests in DNA replication, repair and recombination, as well as teach and mentor students.
Contents
Chapter 1The challenges of maintaining and duplicating the genome
Chapter 2The simple DNA replication system of a bacterial virus
Chapter 3The highly efficient replication system of bacteria
Chapter 4Eukaryotic DNA replication
Chapter 5Replication dynamics — initiating, regulating and terminating cellular DNA replication
Chapter 6Postreplication repair of mismatches and ribonucleotides
Chapter 7DNA topology and the enzymes that alter it
Chapter 8DNA damage — a persistent threat to the genome
Chapter 9Direct reversal of DNA damage
Chapter 10Excision repair — taking advantage of the complementary strand
Chapter 11Repair of double-strand breaks
Chapter 12DNA damage tolerance and translesion DNA polymerases
Chapter 13DNA damage response pathways
Chapter 14DNA replication and repair in human disease
Chapter 15Enzymes of DNA replication and repair fuel modern genomic technologies
About the Author
Kenneth Kreuzer, PhD, is Emeritus Professor of Biochemistry at Duke University. Ken grew up in Western New York, received his Bachelor of Science degree in Biology from MIT (1974) and his PhD in Genetics from the University of Chicago (1978). His graduate research provided some of the earliest evidence that the antibacterial quinolones act by poisoning the enzyme DNA gyrase, and his postdoctoral research at the University of California, San Francisco, identified replication origins of bacteriophage T4. As an independent investigator for three decades at Duke University, Kreuzer and his lab made contributions concerning cytotoxic mechanisms of topoisomerase inhibitors, the role of R-loops in DNA replication, connections between homologous recombination and DNA replication, and mechanisms of double-strand break repair. This research was supported by multiple grants from the NIH and other sources and resulted in over 100 publications. Most of these papers were co-authored by students and postdocs in his lab, who all went on to varied and successful careers of their own.
Ken is a Fellow of the American Academy of Microbiology and of the American Association for the Advancement of Science, and served as Interim Chair of the Duke Biochemistry Department (2007–2010). He has taken a special interest in mentoring and teaching throughout his academic career. He served as Director of the Duke University Summer Research Opportunities Program (1996–2003), the Duke Cell and Molecular Biology Graduate Program (2001–2006), the Duke Post-baccalaureate Research Education Program (PREP; 2003–2008), and as Co-Director of the Duke BioCoRE Program (Initiative to Maximize Student Development; 2013–2015). In honor of his graduate mentoring and diversity efforts, Ken has received the Samuel DuBois Cook Community Betterment Award, the Faculty Award for Graduate Teaching in the Duke Basic Biomedical Sciences, and the Duke University President’s Diversity Award. Since retiring from running an active lab in 2015, Ken and his wife Bev live in the mountains of Southwest Virginia with their dogs and ponies. In addition to writing this book, he has been enjoying woodworking, outdoor activities (golfing, hiking and kayaking), and spending quality time with friends in a beautiful rural setting.
Chapter 1
The challenges of maintaining and duplicating the genome
1.1Introduction
The human genome consists of about 3.3 billion base pairs of DNA spread between the 23 chromosomes. With the exception of the sex chromosomes in males, all the chromosomes are present in two copies, one from each parent, and thus the total sequence length in a human cell is about 6.6 billion base pairs. This DNA sequence complexity determines the precise amino acid sequences of many thousands of proteins, the nucleotide sequence of numerous structural RNAs, and myriad regulatory signals that dictate when genes are turned off and on in response to organism development, nutritional conditions, and stress or disease states. The size of the genome in base pairs can be appreciated by comparing it to the average length of one of the Harry Potter books, roughly 0.7-million alphabet letters. It would take something like 5000 Harry Potter-length books to print out one copy of the information stored in the human genome!
Another way to appreciate the length and complexity of our DNA is to think about all human DNA in your body at any one time. Estimates of the total number of human cell nuclei in an average adult are in the range of 7 trillion.1 The 6.6 billion base pairs of DNA in the human cell nucleus has a length of about 2 m, and so the human DNA in your body would be about 14 trillion (1.4 × 1013) m long if it were lined up end to end. Well, 14 trillion m equals 14 billion (1.4 × 1010) km, and consider that the distance from the earth to the sun is about 0.15 billion (1.5 × 107) km. Your DNA could therefore stretch to the sun and back about 45 times!
In spite of its 6.6-billion base-pair length, the sequence of the genome is passed down to daughter cells with remarkable accuracy in every cell division during a human lifetime. Current estimates of the error rate for this process are in the range of one per billion (109) to one per ten billion base pairs replicated — it sounds even more impressive when this is expressed as an accuracy rate — 99.9999999% of the base pairs are copied correctly (at one error per billion). Comparing again to the Harry Potter books mentioned above, this would be like copying the 5000 imaginary Harry Potter books while making no more than a few mistakes — the vast majority of the 5000 books would be perfect. We will see that the impressive accuracy of genome replication results from a combination of a very accurate DNA replication machine combined with powerful DNA repair pathways, most of which restore the original nucleotide sequence and thereby avoid errors. Furthermore, as we will see, the DNA replication and repair machineries provide a remarkable resistance to DNA damage from both endogenous toxins like oxygen radicals and exogenous sources like the UV in