Browsing by Subject "DNA Replication"
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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. [Keywords: DNA replication, Okazaki fragment RNA primer, S phase, leading strands, laying strands, aging, cancer, telomere overhanger, telomerose]Item Replication-associated base excision repair Of oxidized bases in the mammalian genome(2009-10-31) Corey Allen Theriot; John Papaconstantinou, Ph.D.; Tapas Hazra, Ph.D.; Sankar Mitra, Ph.D.; Isvan Boldogh, Ph.D.; Cornelis Elferink, Ph.D.; Alan Tomkinson, Ph.D.Reactive oxygen species (ROS), the most pervasive endogenous and radiation-induced genotoxic agents induce strand breaks and a plethora of base lesions in DNA that (except double-strand breaks) are repaired via the DNA base excision repair (BER) pathway. Four mammalian DNA glycosylases, namely, OGG1 and NTH1 in the Nth family, and NEIL1 and NEIL2 in the Nei family, with overlapping substrate range initiate BER by excising oxidized base lesions and cleaving the DNA strand. NEIL1 prefers oxidized pyrimidines or ring-opened purines as substrates and is upregulated at the mRNA and protein level during S-phase. NEIL1 also demonstrates the unique able to excise base lesions from forked or single-stranded DNA substrates that mimic intermediates generated during DNA replication. This suggests a direct linkage of NEIL1’s repair activity to genome replication. In addition, inactivating mutations in the NEIL1 gene have been epidemiologically linked with gastric cancer, NEIL1-downregulation induces a mutator phenotype and NEIL1 KO mice display symptoms of the human metabolic syndrome such as obesity, dyslipidemia, and fatty liver disease. These observations lead us to develop the working hypothesis that NEIL1 is involved in a preferential repair pathway for oxidized base damage in the replicating genome where repair of both template strands is equally important because an unrepaired base lesion in either strand could induce mutations. Thus, specific involvement of NEIL1 with the DNA replication machinery may be required to effectively and efficiently accomplish this. In support of our hypothesis, we have identified several new NEIL1 interacting proteins that are components of the DNA replication machinery, including Replication Protein A (RPA), Proliferating Cell Nuclear Antigen (PCNA), Flap Endonuclease 1 (FEN1), DNA Polymerase ä, Replication Factor C (RFC), and DNA Ligase I as well as the stress responsive Rad9-Rad1-Hus1 (9-1-1) DNA sliding clamp. We mapped the overlapping binding sites for all of these interacting protein partners to a small disordered region near the unconserved C-terminus of NEIL1 that is dispensable for its enzymatic activity. In support of the biological significance of these interactions, we showed that the DNA polymerase processivity factor and sliding clamp, PCNA, stimulates NEIL1’s activity on various DNA substrates including forked and single-stranded DNA. We also investigated NEIL1’s association with the DNA damage activated alternative sliding clamp 9-1-1 and showed direct interaction as well as stimulation of NEIL1 activity in a similar fashion as PCNA. In contrast, the RPA complex inhibits NEIL1’s activity when the damage is in the single-stranded region of a DNA primer-template structure, inhibition that is relieved in the presence of PCNA. These results suggest that PCNA and RPA, along with other proteins, collaborate to regulate a replication-associated repair pathway in mammalian cells that not only maintains efficient and proper replication but also repair of oxidative DNA damage to prevent mutagenesis and maintain genomic integrity.Item Telomere Position Effect in Human Cells(2003-04-01) Baur, Joseph Anthony; Shay, JerryTelomeres 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.