Browsing by Subject "DNA Replication"
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Item The Crosstalk Between DNA Mismatch Repair and Replication(2022-05) Zhang, Junqiu; Davis, Anthony John; Castrillon, Diego H.; Erzberger, Jan; Li, Guo-MinDNA replication fidelity relies on DNA mismatch repair (MMR) and the proofreading nuclease activity of DNA polymerases. Normally, biosynthetic errors can be removed by the polymerase's proofreading nuclease activity upon their incorporation, and those errors that have escaped the proofreading nuclease are corrected by MMR. However, this model is challenged by the fact that cells expressing a proofreading-deficient P286R polymerase ɛ (Polɛ-P286R) display a hypermutable phenotype usually seen in MMR-deficient cells, implying the blockage of MMR function by Polɛ-P286R. We show here that consistent with frequent misincorporation by Polɛ-P286R, elevated levels of MMR proteins were found in replicating DNA/chromatin in Polɛ-P286R cells, but this does not result in a reduced mutation frequency, suggesting that cluster binding of MMR proteins at the replication fork inhibits MMR. Instead, the high-level binding of MMR proteins blocks the recruitment of fork protection factors FANCD2 and BRCA1 to replication forks, and promotes MRE11-catalyzed nascent strand degradation. This MMR-dependent degradation causes DNA breaks and chromosome abnormalities, thereby promoting an ultramutator phenotype. Therefore, our findings identify a novel MMR function in triggering replication stress response to promote genome instability when replication forks are filled with biosynthetic errors. The importance of MMR in maintaining genome stability prompts us to further study the mechanism of MMR in vitro, particularly how the MMR initiation complex is formed in response to misincorporation. Using purified recombinant proteins, we assembled MMR initiation complex in vitro and visualized protein-protein and protein-DNA interactions under transmission electron microscopy. These analyses allowed us to gain molecular insights into the mechanism of MMR initiation.Item Defining Multiple Steps in Human Telomere End Processing(2012-07-10) Chow, Tan Hoi Tracy; Shay, Jerry W.Telomere overhangs are essential for chromosome end protection and telomerase extension, but how telomere overhangs are generated is unknown. Due to the classic end replication problem, leading DNA daughter strands are initially blunt while lagging daughters are shorter by at least the size of the final RNA primer, which historically is believed to be located at extreme chromosome ends. We developed a variety of new approaches to define the steps in the processing of these overhangs. Understanding the number and nature of the overhang processing events is crucial in establishing the roles of candidate proteins involved. We here define these steps in normal human cells. We show the final lagging RNA primer is positioned ~70-100 nt from chromosome ends (not at the extreme ends), and is not removed for ~1hr following replication. Therefore, the location of the RNA primer, rather than its size, is a primary driving force for telomere shortening. Moreover, we demonstrate that telomere end-processing occurs in two distinct phases following telomere duplex replication. During the early phase, which occupies 1-2 hours following telomere replication, several steps occur on both leading and lagging daughters. Leading telomere processing remains incomplete until late S/G2 when the C-terminal nucleotide is specified, referred to as the late phase. Furthermore, in human cancer cells under maintenance condition, telomerase extension is uncoupled from C-strand fill-in. These results uncover crucial mechanistic details of the DNA end-replication problem as well as several specific steps in telomere overhang processing. These results also indicate the presence of previously unsuspected complexes and signaling events required for the replication of the ends of human chromosomes. The findings and the methods developed will now provide the basis for examining candidate factors that may function to regulate particular steps in telomere length homeostasis with implications in both cellular aging and cancer.Item Discovering GCNA: A Novel Regulator of Germline Genomic Stability(2018-10-15) Bhargava, Varsha; Mendell, Joshua T.; Buszczak, Michael; Olson, Eric N.; Tu, BenjaminGerm cells transfer genetic information across generations. Any change in germ line DNA is inherited by succeeding generations. Therefore, germ cell DNA must be protected from both internal and external assault. An advantage of sexual reproduction stems from the ability to generate variation by exchange of chromosomal segments during meiosis. During meiosis, hundreds of double-stranded DNA breaks are initiated at once, which if generated in most other cell types would introduce chromosomal aberrations. Germ cells, however, execute the formation of these breaks while preventing their deleterious effects from becoming pervasive throughout the genome. The mechanisms underlying the robustness of germ cells in the face of DNA damage, however, are poorly understood. We initiated an in vivo CRISPR-Cas9 knockout screen for genes highly enriched in the Drosophila female germ line. From this screen, we identified Germ Cell Nuclear Acidic Peptidase (GCNA) as a conserved regulator of genome stability across multiple species. Loss of GCNA results in replication stress, chromosomal instability, and an accumulation of DNA-protein crosslinks (DPCs). Disruption of GCNA leads to an accumulation of nuclear Top2 and Top2 DPCs. This work shows GCNA protects germ cells from damage and provides novel insights into the conserved networks that promote genome integrity across generations.Item GCNA: Guardian of the Genome(2020-05-01T05:00:00.000Z) Goldstein, Courtney DaVee; Abrams, John M.; Buszczak, Michael; Brekken, Rolf A.; Olson, Eric N.The propagation of species depends on the ability of germ cells to protect their genome in the face of numerous exogenous and endogenous threats. While germ cells employ a number of know repair pathways, specialized mechanisms that ensure high-fidelity replication, chromosome segregation, and repair of germ cell genomes remain incompletely understood. Here, we identify Germ cell nuclear acidic peptidase (GCNA) as a conserved regulator of genome stability in flies, worms, zebrafish and human germ cell tumors. GCNA contains an acidic intrinsically disordered region (IDR) and a protease-like SprT domain. In addition to chromosomal instability and replication stress, Gcna mutants accumulate DNA-protein crosslinks (DPCs). GCNA acts in parallel with a second SprT domain protein Spartan. Structural analysis reveals that while the SprT domain is needed to limit meiotic and replicative damage, much of GCNA's function maps to its IDR. This work shows GCNA protects germ cells from various sources of damage, providing novel insights into conserved mechanisms that promote genome integrity across generations.Item [Southwestern News](1996-01-07) McNeill, Bridgette RoseItem Telomere Position Effect in Human Cells(2003-04-01) Baur, Joseph Anthony; Shay, Jerry W.Telomeres are tracts of repetitive DNA that cap the ends of linear chromosomes. Each time the chromosome is duplicated, a small amount of telomeric DNA is lost from the end due to factors inherent in the mechanism of DNA replication. The result is a net shortening of telomeres with each cell division, unless new repeats are synthesized through the action of the enzyme telomerase. Most human somatic cells lack telomerase activity and so continued cell division leads to telomere shortening. After a limited number of divisions (the "Hayflick limit"), it is believed that a few critically shortened telomeres trigger a state of growth arrest termed replicative senescence.