The Effect of Damaged Bases on The End Joining of DNA Double Strand Break Ends

DNA double strand breaks (DSBs ) are extremely toxic to cells because they can lead to genomic rearrangements and even cell death. Two main pathways can repair DSBs: the homologous recombination repair (HRR) pathway and the non-homologous end-joining (NHEJ) pathway. NHEJ is the primary repair pathway in mammalian cells. HRR repairs single strand breaks (SSBs) or DSBs, mostly during late S phase and G2 phase of the cell cycle, by using an undamaged copy of the DNA sequence, and is therefore largely error-free, while the NHEJ pathway repairs DSBs without the requirement for sequence homology, may be error-free or error-prone, and is most active during G1 phase. Thymine glycol (Tg), the most common oxidation product of thymine. It produced endogenously as a consequence of aerobic metabolism or via exogenous factors such as ionizing radiation (IR), it is one of the predominant types of base modifications produced by ionizing radiation. Due to clustering of radiation – induced ionizations, Many DSBs induced by ionizing radiation bear damaged bases, including (Tg) moieties at or near the DSB ends that may interfere with subsequent gap filling and ligation. Artemis is a nuclease that is involved in the processing of termini during repair of DSBs and in modifying termini of complex DSBs. It has 5′–3′ exonuclease activity specific for single- stranded DNA, but, in the presence of DNA-PK, Artemis demonstrates endonuclease activity that is utilized in the removal of 3′ phosphoglycolate termini and 5′ overhangs, in the shortening of 3′ overhangs at DSBs, and in the opening of hairpin ends. To assess the ability of NHEJ to rejoin DSBs accompanied by Tg lesions and to elucidate the aspects of the possible role of Artemis in DSB repair, linearized plasmids with Tg either at the 3’ terminus of a blunt end (designated Tg1) or three or two bases from the end (Tg3 and Tg2, respectively), were subjected to a repair assay using XRCC4-like factor (XLF) deficient cell extracts, with or without the addition of XLF and/or Artemis, EndoIII and ddTTP. The data indicated that, the cell extract could ligate the plasmids with Tg1 and Tg2 with extremely low efficiency but could repair plasmid with Tg3 as efficiently as unmodified plasmid. In addition, Plasmids with Tg1and Tg2 were treated with Endonuclease III and ddTTP to test whether the end joining occurred before or after Tg removing, neither one had any effects on plasmids with Tg1. However, plasmids with Tg2 showed reduced in the intensity upon treatment with Endonucleases III and ddTTP, which suggested some ligation occur while Tg still present. In Artemis reaction, substrate with Tg2 and Tg3 could stimulate Artemis mediated trimming but not Tg1. Addition of EndoIII or ddTTP to plasmid with Tg3 resulted in a significant decrease in the intensities of the bandsrepresenting ligated products compared to XLF alone, suggesting that in some of the ligated products Tg are still present, while in others Tg had been removed and replaced by polymerization with normal nucleotides. Taken together, our results indicated that cell extract could ligate the plasmid with Tg located at the third base to DSB with high efficiency compared to plasmids with Tg1 and Tg2 which apparently this ability was severely inhibited when it located at or in the second position to DSB ends. Moreover, Artemis is also capable of trimming of thymine glycol at the second or third position from DSB ends with limited capability but inhibited by the presence of thymine glycol at the break site

Rejoining of DNA Double-Strand Breaks and Genome Stability: From Host-Pathogen Interactions to Break-Induced Mutagensis.

An essential task for cell survival is the maintenance of genome stability despite various environmental and physiological stresses. These stresses, such as UV light and ionizing radiation, often damage DNA and result in various types of DNA lesions. If not properly resolved, DNA lesions in humans can cause autoimmune deficiency, neurodegenerative disorders and cancer. Among these lesions, DNA double-strand breaks (DSBs) are one of the most cytotoxic and greatly threaten the integrity of the genome. In addition, DSBs may act as potential hotspots for genomic integration of exogenous DNA fragments, such as the transfer DNA (T-DNA) delivered by the plant pathogen Agrobacterium. DSBs can be repaired by religation of two broken ends by DNA ligase IV via the nonhomologous end joining (NHEJ) pathway, of which the repair fidelity can be compromised by diverse break structures, resulting in mutagenesis. I sought to further understand the complex contribution of NHEJ to genome stability in research projects conducted using both Agrobacterium-mediated plant transformation and Saccharomyces cerevisiae as model systems. My first project examined the process of double-stranded T-DNA formation using functional assays and demonstrated that annealing of synthetic oligonucleotides to the single-stranded T-DNA can initiate such process in plant cells. A second project developed an Agrobacterium-mediated transformation vector system, in which multiple expression cassettes can be assembled on a single vector using zinc finger nucleases (ZFNs) and homing endonucleases, facilitating delivery of multiple genes in plants. A third project investigated the consequences of having a catalytically inactive DNA ligase IV in yeast NHEJ and led to discovery of an imprecise NHEJ pathway mediated by ligase Cdc9. A last project studied the impact of overhang polarity of chromosomal DSBs on the kinetics and fidelity of yeast NHEJ and results suggest that 5’ overhanging DSBs can cause more frequent mutagenesis despite more efficient rejoining as compared to 3’ overhanging DSBs. Collectively, my dissertation research provides new evidence of the mechanisms governing the important process of double-stranded T-DNA formation during plant genetic transformation, as well as new insights into NHEJ mutagenesis which could lead to different human diseases.PHDMolecular, Cellular and Developmental BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/111334/1/zbliang_1.pd

Recombinant antibodies for the study of livestock infection: from basic genetics to single-chain Fvs

Molecular biology has provided new opportunities to understand better the functioning of the immune system and to exploit this information for the construction of specific antibodies against a wide variety of antigens including the pathogens of humans and animals. In spite of the economic importance of cattle, many aspects of the immunology of this animal remain uncharacterised and tools to understand better bovine infections are lacking. This project has addressed aspects of both issues. The bovine immunoglobulin (Ig) system resembles that of other domesticated mammals in some respects, but other properties (eg the length of the third antigen-binding region of the heavy chain) appear unique. The first area for investigation in this project was to characterise the bovine JH locus and to understand why Ig rearrangement apparently favours a single JH segment. PCR was used to recover JH sequence from genomic DNA, either from non-lymphoid tissues or lambda vectors isolated and studied by other investigators. A region of 3200 bp was characterised which included the DQ52 segment, 6 JH segments and the heavy chain enhancer. The bovine DQ52 sequence is longer than those of other species and differs in sequence from a common consensus. For the most part, the JH locus is homologous to that of the sheep. The sixth JH segment identified appears to undergo rearrangement and is expressed in a minority of cattle antibodies. However, none of the segments carried the sequence which is most commonly expressed in bovine Ig. To identify which segment participates in this process, sequence was recovered from the rearranged genomic DNA of isolated bovine B cells using PCR with primers against VH and JH regions. This implicated the rearrangement of the fourth JH segment in the formation of bovine Igs but as the sequence differed between germline and rearranged copies, it appears that a non-conventional process operates in cattle. It is proposed that a gene conversion event or modified rearrangement process introduces sequence to form the fourth framework region of bovine Ig which does not exist at the JH locus in the germline. The mechanism of this modification requires more investigation. The second part of this project aimed to construct a library of recombinant bovine Fab antibodies from the Ig repertoire of a calf vaccinated against Mannheimia haemolytica (previously named Pasteurella haemolyticd)

DNA Damage Induction by Bilirubin Induced Oxidative Stress and Activation of DNA Repair Pathways by Bilirubin

“Kernicterus” due to hyperbilirubinemia is one of main cause of irreversible brain damage in low and middle income countries. Deaths due to “kernicterus” are ranked within the top 3 causes of neonatal death in African countries. Neonates experiencing permanent or temporary absence or reduced activity of Uridine diphosphate-glucuronosyl-transferase A1 (UGT1A1) enzyme present increased levels of total unconjugated bilirubin (UCB) in the blood. When the bilirubin binding capacity of serum albumin is saturated, this results in the increase in the free fraction of bilirubin (Bf). The excess of Bf is accumulated in lipid-rich tissues such as the brain, where it may reach toxic levels causing bilirubin-induced encephalopathy in jaundiced newborns and patients with Crigler-Najjar syndrome Type I, leading to kernicterus if not promptly treated. Bilirubin causes severe neurological dysfunction by affecting several different cellular pathways such as, induction of oxidative stress, endoplasmic reticulum (ER) stress, autophagy, neuroinflammation and DNA damage. The work done in this thesis focus on the DNA damaging effect of bilirubin on neuronal (SH SY 5Y) and non-neuronal (HeLa) cells and the effect of bilirubin on the different double stranded break (DSB) repair pathways was investigated using HeLa DR GFP and HeLa cell lines. Bilirubin exposure led to time-dependent increase in DNA damage in neuronal cells. Treatment with the anti-oxidant N-acetylcysteine (NAC) caused a dose-dependent decrease in DNA damage, suggesting a key role of oxidative stress in bilirubin-induced DNA damage. In the second part of this work, I studied the effect of bilirubin on different DSB repair pathways using HeLa cell lines. Treatment with bilirubin modulated Homologous Recombination (HR) in a time-dependent manner. In fact, treatment with different doses of bilirubin led to a dose-dependent increase in HR. Bilirubin-induced increase in HR was reversed by addition of NAC, suggesting the important role of oxidative stress in bilirubin-induced modulation of HR. DNA damage analysis after bilirubin treatment showed an increase in DNA damage in HeLa cells. Unexpectedly, bilirubin-induced DNA damage had no effect on the cell cycle profile. Similarly, exposure to bilirubin concentrations similar to those found in patients led to an increase in the Non-Homologous End Joining (NHEJ) pathway. These results suggest the general increase in DSB repair pathways by bilirubin

Double-Strand Break Repair by Non-homologous End-Joining

Non-homologous end-joining (NHEJ) is an important mechanism of double-strand break repair (DSBR) which is largely conserved from yeast to humans. Mammalian NHEJ is dependent on the Ku, DNA-PKcs, XRCC4 and DNA ligase IV proteins. NHEJ-defective rodent cells are highly sensitive to ionising radiation, implicating NHEJ as a critical mechanism for the repair of radiation-induced double-strand breaks (DSBs). DNA termini at sites of radiation-induced DSBs exhibit modifications, including the presence of 5'-hydroxyl and 3'-phosphate or phosphoglycolate groups. During DSBR, these termini must undergo enzymatic processing to return the normal 5'-phosphate and 3'-hydroxyl groups required for ligation. Despite the activities of the core NHEJ proteins being well documented, little is known about the auxiliary processing factors required for repair of ionising radiation-induced breaks. Using an in vitro DNA end-joining assay catalysed by human cell-free extracts I have investigated the wider protein requirements of NHEJ and the ability of extracts to join certain modified DNA termini. In contrast to the essential role for the Mre11 and Xrs2 proteins in the joining of complementary termini in Saccharomyces cerevisiae, the human homologues, MRE11 and NBS1 were not required for the joining of complementary protruding 5' termini. Joining of modified and non-complementary termini implicated requirements for exonuclease and polymerase activities during extract-catalysed end-joining. Moreover, end-joining reactions with DNA molecules containing 5'-hydroxyl termini demonstrated a role for polynucleotide kinase (PNK) in the repair of radiation-induced breaks. The defective end-joining observed with PNK-depleted extracts was complemented by addition of physiological amounts of purified human PNK, but not T4 PNK. In addition, phosphorylation of 5'-hydroxy termini was not observed when DNA end-joining was blocked, either by use of XRCC4 antibodies or DNA-PKcs-defective extracts, implicating co-ordination between the phosphorylation and end-joining reactions. Finally, gel filtration and coimmunoprecipitation experiments indicated that PNK might be associated with the NHEJ proteins in vivo

Thermodynamics of DNA binding and break repair by the Pol I DNA polymerases from Escherichia coli and Thermus aquaticus

Klenow and Klentaq are the “large fragments” of the Pol I DNA polymerases from Escherichia coli and Thermus aquaticus. Examination of the DNA binding thermodynamics of both polymerases to replication versus repair substrates shows that Klenow binds primed-template DNA with up to 50X higher affinity than it binds to a nicked DNA, gapped DNAs, DNA with blunt-end or a 3’ overhang, while Klentaq binds all of these DNAs similarly. The presence of 5’ or 3’ phosphates has slightly different effects on DNA binding by both polymerases. In contrast, both polymerases bind mismatched DNA tighter than matched DNA, suggesting that they may share a similar mechanism to identify mismatched DNA, despite the lack of proofreading ability in Klentaq. The effects of Klenow and Klentaq on ligation of DNA ligase were also studied. Both polymerases stimulate the intermolecular ligation activity of E. coli DNA ligase at concentrations sub-stoichiometric to the DNA concentration. This effect occurs with E. coli DNA ligase, but not for T4 and Taq ligases. Additionally, neither polymerase significantly enhances ligation of a substrate containing a single nick, suggesting that the polymerases bridge the two DNA ends during intermolecular ligation. The nucleotide incorporation activities of both polymerases on substrates minicing double-strand breaks (DSBs) were also examined. Both proteins are able to “repair” DSBs via alignment-based strand-displacement DNA synthesis. Moreover, their repair abilities have different dependences on 5’ phosphate and DNA ligase when DSBs contain non-cohesive ends. Additionally, both proteins mediated palindrome amplification alone when the short inverted repeats occur near DNA breaks, suggesting that short inverted repeats in prokaryotes may help in DSB repair. 5’ phosphate at the matched break end is required for DSBs repair by both polymerases when one break end contains 3 consecutive mismatches. Results of the electrophoretic mobility shift assay show that Klenow-DNA complexes are observed as slow or fast moving bands, or both while all Klentaq-DNA complexes are observed as slow moving bands. The protection of both ends of a DNA by Klenow from exonuclease digestion suggests that the slow moving bands may correspond to the 2:1 polymerase-DNA complex

RESOLUTION OF PROXIMAL OXIDATIVE BASE DAMAGE AND 3′-PHOSPHATE TERMINI FOR NONHOMOLOGOUS END JOINING OF FREE RADICAL-MEDIATED DNA DOUBLE-STRAND BREAKS

Clustered damage to DNA is a signature mark of radiation-induced damage, which involves damage to the nucleobases and/or DNA backbone. Double-strand breaks created by damaging agents are detrimental to cell survival leading to chromosomal translocations. Normal cells employ Non-homologous end-joining because of its faster kinetics, to suppress chromosomal translocations. However, the presence of complex DNA ends constitutes a significant challenge to NHEJ. Location of Thymine glycol (Tg) at DSB ends was a potential hindrance to end joining. The substrate with Tg at the third position (Tg3) from the DSB joined better than when present at the fifth position (Tg5). However, hNTH1 assay showed Tg5 to be a better substrate than Tg3 for BER, potentially explaining the increased Tg removal and decreased end joining of Tg5 in extracts. Nonetheless, there appeared to be no preference in the susceptibility of 5’-Tg substrates with Tg at the second and third positions from DSB ends. Polynucleotide kinase phosphatase is crucial in restoring the 3′ hydroxyl, and 5′ phosphate ends at strand breaks. No other enzyme is known to possess PNKP’s activity in mammalian cells at DSBs. Experiments done with PNKP knockout cells have shown some activity similar to PNKP, which appeared to be a part of NHEJ and was not pharmacologically inhibited by PNKP inhibitor. Additionally, core NHEJ factors XRCC4 and XLF influenced the activities of PNKP. Overall, these experiments suggest that Tg repair is dependent on the position from DSB and an alternative enzyme processes 3′- PO, and 5′-OH ends