When a replication fork encounters a DNA lesion in a template strand, replication gives way to DNA repair and recombination. These encounters define an interface in DNA metabolism that gives rise to much of the DNA rearrangement, repeat expansion, and mutagenesis that defines genome instability. This is ultimately manifested in tumor evolution in eukaryotes and the development of antibiotic resistance and increased pathogenicity in bacteria. Recent investigation of events that occur at the fork have largely overlooked an important genomic venue for repair ? lesions left behind the fork in postreplicative gaps. The existence of these gaps has been appreciated for over 5 decades, but progress has been limited by methodology that has been inadequate to properly explore their general importance and repair. These gaps are primary substrates for DNA synthesis by translesion DNA polymerases, recombinational DNA repair, and replicational template switching, all processes linked with genomic instability. This proposal frames a multidisciplinary effort to explore how postreplicative gaps are generated, how often they are formed, what circumstances trigger formation, what occurs within them, and how the various paths of gap repair are prioritized and governed. The work is an outgrowth of advances in understanding three enigmatic protein activities, RarA, Uup, and RadD. RarA, an AAA+ ATPase that functions at the replisome to generate postreplicative gaps on the lagging strand, is one key. To tackle this problem, we bring together world-class expertise in biochemistry, genetics, molecular biology, and biophysics. We will develop new methods, including novel single-molecule approaches towards detecting and quantifying gaps, and characterizing the proteins acting on them. While driven by our mechanistic questions, these methods will broadly benefit research in genomic maintenance. The five specific aims constitute a systematic attack on the problem. The mechanism of RarA protein is the focus of Aim 1.
Aim 2 provides the first effort to quantify gap formation in vivo and determine the factors that trigger gap formation.
Aim 3 explores the functions of Uup and RadD, two enzymes that suppress template switching within gaps.
Aim 4 explores how various pathways of gap repair are organized and governed. The proposal is completed with Aim 5, an exploration of translesion DNA polymerases acting within gaps, a key source of genomic mutagenesis.

Public Health Relevance

Genomic instability gives rise to aging, tumor evolution, DNA repeat expansion associated with multiple disease states, the development of antibiotic resistance in bacteria, and a general reduction in evolutionary fitness. Much genome instability is associated with the interfaces between DNA replication, repair, and recombination that become prominent when replication forks encounter DNA damage. This proposal addresses the molecular basis of an essential yet too often overlooked source of genomic instability, the repair of lesions in postreplicative gaps that are left behind the replication fork. As gap repair is a primary source of mutagenesis and genomic rearrangements, we are proposing an integrated strategy to attain an in-depth understanding of gap formation and repair, aimed at providing new avenues to slow tumor growth and bacterial antibiotic resistance.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project with Complex Structure (RM1)
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Special Emphasis Panel (ZRG1)
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Reddy, Michael K
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University of Wisconsin Madison
Earth Sciences/Resources
United States
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