Fanconi anemia (FA) is a human genetic disease that is characterized by bone marrow failure, developmental abnormalities, and a high incidence of leukemias and solid tumors. At the cellular level, the distinguishing feature of FA is extreme sensitivity to chemicals that induce DNA interstrand cross-links (ICLs). FA is caused by defects in thirteen Fanc proteins, eight of which form a ubiquitin ligase that mono-ubiquitylates the FancI-FancD2 heterodimer (the """"""""ID"""""""" complex). Indirect evidence strongly indicates that these FA proteins promote ICL repair, but their specific role remains enigmantic, in large part due to the absence of molecular assays that directly measure ICL repair. Using Xenopus egg extracts, we have developed the first experimental system that supports FA pathway-dependent ICL repair in vitro. We show that repair is intimately coupled to DNA replication. Specifically, repair involves convergence of two DNA replication forks on the ICL. After initially pausing 20 nucleotides from the ICL, the 3'end of one leading strand advances to the lesion (Approach), inserts a nucleotide across from the damaged base (translesion DNA synthesis or TLS), and is then extended beyond the lesion in a DNA polymerase 6-dependent fashion (Extension). Concurrent with lesion bypass, nucleolytic incisions occur on the other template strand. Ultimately, a significant fraction of the input DNA is fully repaired in a replication-dependent manner. Importantly, immunodepletion of FancD2 from egg extracts dramatically reduces repair and arrests lesion bypass immediately prior to the TLS step. The development of this cell-free system represent an experimental breakthrough that allows a systematic analysis of the factors involved in ICL repair, including the FA proteins. In this grant, we focus on the role of the FA proteins in lesion bypass.
In Specific Aim 1, we address how the Approach step is accomplished, including whether it depends on FancM or FancJ.
In Aim 2, we address which polymerases promote the translesion DNA synthesis step, so that we may, in Aim 3, elucidate how the ID complex catalyzes this event. The experiments will elucidate the molecular defects that underlie Fanconi anemia.
Fanconi anemia (FA) is a cancer predisposition disorder which is caused by defects in a set of proteins that are thought to fix DNA inter-strand cross-links (ICLs), a particularly dangerous type of DNA damage. However, the mechanism by which the FA proteins normally fix ICLs is obscure. We have discovered that extracts derived from frog eggs recapitulate the process of ICL repair in a cell-free environment. We will use this system to elucidate the function of FA proteins in ICL repair.
|Sparks, Justin L; Chistol, Gheorghe; Gao, Alan O et al. (2018) The CMG Helicase Bypasses DNA-Protein Cross-Links to Facilitate Their Repair. Cell :|
|Larsen, Nicolai B; Gao, Alan O; Sparks, Justin L et al. (2018) Replication-Coupled DNA-Protein Crosslink Repair by SPRTN and the Proteasome in Xenopus Egg Extracts. Mol Cell :|
|Amunugama, Ravindra; Willcox, Smaranda; Wu, R Alex et al. (2018) Replication Fork Reversal during DNA Interstrand Crosslink Repair Requires CMG Unloading. Cell Rep 23:3419-3428|
|Semlow, Daniel R; Zhang, Jieqiong; Budzowska, Magda et al. (2016) Replication-Dependent Unhooking of DNA Interstrand Cross-Links by the NEIL3 Glycosylase. Cell 167:498-511.e14|
|Räschle, Markus; Smeenk, Godelieve; Hansen, Rebecca K et al. (2015) DNA repair. Proteomics reveals dynamic assembly of repair complexes during bypass of DNA cross-links. Science 348:1253671|
|Zhang, Jieqiong; Dewar, James M; Budzowska, Magda et al. (2015) DNA interstrand cross-link repair requires replication-fork convergence. Nat Struct Mol Biol 22:242-7|
|Budzowska, Magda; Graham, Thomas G W; Sobeck, Alexandra et al. (2015) Regulation of the Rev1-pol ? complex during bypass of a DNA interstrand cross-link. EMBO J 34:1971-85|
|Duxin, Julien P; Walter, Johannes C (2015) What is the DNA repair defect underlying Fanconi anemia? Curr Opin Cell Biol 37:49-60|
|Long, David T; Joukov, Vladimir; Budzowska, Magda et al. (2014) BRCA1 promotes unloading of the CMG helicase from a stalled DNA replication fork. Mol Cell 56:174-85|
|Duxin, Julien P; Dewar, James M; Yardimci, Hasan et al. (2014) Repair of a DNA-protein crosslink by replication-coupled proteolysis. Cell 159:346-57|
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