Download i REPAIR OF BIPYRIMIDINE PHOTOPRODUCTS AT TELOMERES OF ULTRAVIOLET LIGHT ... PDF

Titlei REPAIR OF BIPYRIMIDINE PHOTOPRODUCTS AT TELOMERES OF ULTRAVIOLET LIGHT ...
LanguageEnglish
File Size1.9 MB
Total Pages133
Table of Contents
                            TITLE PAGE
COMMITTEE MEMBERS
ABSTRACT
TABLE OF CONTENTS
LIST OF TABLES
	Table 1. Oligonucleotides used in the study
LIST OF FIGURES
	Figure 1: Telomere models
	Figure 2: DNA damage and repair pathways
	Figure 3: Electromagnetic light spectrum
	Figure 4: Structures of ultraviolet light induced CPDs and 6-4 PPs
	Figure 5: Sequence of events in eukaryotic nucleotide excision repair (NER) pathway
	Figure 6: Telomere isolation assay
	Figure 7: Restriction enzyme digestion of genomic DNA
	Figure 8: Quantification of CPD formation and removal in telomeres from UVC exposed BJ-hTERT cells
	Figure 9: UVC sensitivity and proliferation of BJ-hTERT and XP-A cells
	Figure 10: Quantification of 6-4 PP formation and removal in telomeres from UVC exposed BJ-hTERT cells
	Figure 11: Estimation of CPDs and 6-4 PPs in BJ-hTERT genomic DNA after UVC exposure
	Figure 12: XPA protein is required for 6-4 PP removal from telomeres
	Figure 13: Quantification of CPD formation and removal in genomic DNAfrom UVC exposed XP-A cells
	Figure 14: Quantification of 6-4 PPs formation and removal in telomericDNA from UVC exposed U2OS cells
	Figure 15: Quantification of CPD formation and removal in genomic DNA from UVC exposed U2OS cells
	Figure 16: A cyclobutane pyrimidine dimer inhibits TRF1 binding totelomeric DNA
	Figure 17: UVC induces sensitivity and inhibits proliferation of XPAC cells
	Figure 18: UVC induces telomere aberrations in XPAC and XP-A cells
	Figure 19: Quantification of CPD and 6-4 PP formation and removal in genomic DNA from UVC exposed XPAC cells
	Figure 20: Western blotting analysis of BJ-hTERT, XP-A and XPAC cells for the presence of XPA protein
	Figure 21: Inhibition of XPF-ERCC1 activity on stem-loop substrate by TRF2
ACKNOWLEDGEMENTS
ABBREVIATIONS
1 INTRODUCTION
	1.1 TELOMERES: AN OVERVIEW
		1.1.1 Beginning of the end
		1.1.2 A solution to the end replication problem
		1.1.3 Electron microscopy examination of telomeres and the discovery of telomere looping
		1.1.4 Current paradigm of telomeres with shelterin and associated proteins
		1.1.5 Role of shelterin in preserving telomere integrity
		1.1.6 Telomere length maintenance mechanisms and consequences of telomere dysfunction
	1.2 DNA DAMAGE AND REPAIR
		1.2.1 Ultraviolet light (UV) as a toxic agent
		1.2.2 Oxidative base damage in DNA
		1.2.3 UV-induced bulky lesions and repair in genomic DNA
		1.2.4 UV mutagenesis and photoproduct removal
		1.2.5 Types of Nucleotide Excision Repair (NER)
		1.2.6 The case of no repair: XP and complications arising from lack of NER
		1.2.7 UV damage and NER at telomeres
	1.3 STATEMENT OF THE PROBLEM AND HYPOTHESIS
	1.4 STATEMENT OF PUBLIC HEALTH SIGNIFICANCE
2 TELOMERES ARE PROFICIENT IN REMOVAL OF UV INDUCED PHOTOPRODUCTS VIA NUCLEOTIDE EXCISION REPAIR
	2.1 ABSTRACT
	2.2 SIGNIFICANCE
	2.3 INTRODUCTION
	2.4 MATERIALS AND METHODS
		2.4.1 Gel shift assays
		2.4.2 Cell culture and exposures
		2.4.3 Cell viability and proliferation assays
		2.4.4 Genomic DNA and telomere purification
		2.4.5 Telomere restriction fragment analysis
		2.4.6 Immuno-spot blot detection of DNA photoproducts
		2.4.7 Quantitative PCR detection of DNA photoproducts
		2.4.8 Statistical analysis
	2.5 RESULTS
		2.5.1 Purification of telomeres from human cells
		2.5.2 BJ-hTERT telomeres exhibit formation and removal of CPDs and 6-4 PPs
		2.5.3 Removal of 6-4 PPs at telomeres depends on XPA protein
		2.5.4 An unrepaired cyclobutane pyrimidine dimer inhibits TRF1 binding
	2.6 DISCUSSION
	2.7 BIOLOGICAL IMPLICATIONS
3 INVESTIGATING ROLES FOR NUCLEOTIDE EXCISION REPAIR IN PROTECTING TELOMERES FROM DEFECTS INDUCED BY UV IRRADIATION
	3.1 INTRODUCTION
	3.2 MATERIALS AND METHODS
		3.2.1 Cell culture and exposures
		3.2.2 Cell viability and proliferation assays
		3.2.3 Telomere fluorescent in Situ hybridization assays
		3.2.4 Genomic DNA purification and immune-spot blot detection of DNA photoproducts
		3.2.5 Western blotting for XPA protein
		3.2.6 Statistical methods
	3.3 RESULTS
		3.3.1 UVC sensitivity of XPAC cells compared to XP-A cells
		3.3.2 UVC causes an increase in telomere aberrations in XP-A and XPAC cells
		3.3.3 CPDs and 6-4 PPs are poorly removed from genomic DNA of XPAC cells
		3.3.4 XPAC cells express abnormally high levels of XPA protein
		3.3.5 Discussion
4 FINAL DISCUSSION
	4.1 SUMMARY OF FINDINGS
	4.2 LIMITATIONS OF THE ASSAY
	4.3 FUTURE DIRECTIONS
	4.4 BIOLOGICAL IMPLICATIONS
	4.5 STUDY CONCLUSIONS
APPENDIX: PRELIMINARY STUDY ON NER AND SHELTERIN INTERACTIONS
	SHELTERIN INHIBITS NER PROTEIN XPF-ERCC1 CLEAVAGE OF A STEM LOOP SUBSTRATE
BIBLIOGRAPHY
                        
Document Text Contents
Page 1

i



REPAIR OF BIPYRIMIDINE PHOTOPRODUCTS AT TELOMERES OF

ULTRAVIOLET LIGHT IRRADIATED MAMMALIAN CELLS









by

Dhvani Parikh



MS, Gujarat University, India, 2006

MS, Environmental Health Sciences, New York University, 2009










Submitted to the Graduate Faculty of

Graduate School of Public Health in partial fulfillment

Of the requirements for the degree of

Doctor of Philosophy









University of Pittsburgh

2014

Page 2

ii

UNIVERSITY OF PITTSBURGH

Graduate School of Public Health

This dissertation was presented

By

Dhvani Parikh

It was defended on

November 12, 2014

And approved by

Chairperson: Aaron Barchowsky, PhD, Professor, Department of Environmental
and Occupational Health, Graduate School of Public Health, University of Pittsburgh

Ben Van Houten, PhD, Professor, Department of Pharmacology and Chemical
Biology, School of Medicine, University of Pittsburgh

Peter Di, PhD, Assistant Professor, Department of Environmental and Occupational
Health, Graduate School of Public Health, University of Pittsburgh

Dissertation Advisor: Patricia Lynn Opresko, PhD, Associate Professor,
Department of Environmental and Occupational Health, Graduate School of Public

Health, University of Pittsburgh

Page 66

50



(A) Cells were exposed to 10 J/m2 UVC, incubated in fresh media, and then counted
after each recovery time point (0-72 h). Cell counts were normalized to the 0 h and
plotted against recovery time. Values and error bars are from means and SE from three
independent experiments. (B) Cells were exposed to 10 J/m2 UVC, incubated in fresh
media, and then harvested after each recovery time point (0-48 h) by trypsinization after
washing. Cells were counted manually on a hemocytometer for total cells and Trypan
blue positive dead cells. Percent viability was calculated as described in Materials and
Methods. Values represent the mean and SE from three independent experiments. (C)
Cells were exposed to 0, 5, or 10 J/m2 UVC, recovered for 6h, sub-cultured and counted
after 7 days of incubation. Survival was calculated as percent of untreated and plotted
against UVC dose. Values and error bars are means and SE from three independent
experiments.


Figure 9: UVC sensitivity and proliferation of BJ-hTERT and XP-A cells

Page 67

51

The other common photoproduct 6-4 PP had not been previously examined at telomeres.

6-4 PP lesions induce greater distortion in the duplex DNA and are repaired more rapidly

than CPDs, however, they are formed at a lower frequency [131, 132]. The 10 J/m2 UVC

exposure generated about 1.4 6-4 PPs per 10 kb of genomic DNA (Fig. 11). Therefore,

higher amounts of loaded purified telomeres (15 ng isolated from 200 g genomic DNA)

were required for reliable 6-4 PP detection. Following 10 J/m2 UVC, the 6-4 PP signal

was on average 1.9-fold ( 0.32) lower for bulk telomeric DNA, compared to equal

amounts (15 ng) of bulk genomic DNA (Fig. 10A). This equates to approximately 0.74 6-

4 PPs/10 kb telomeric DNA, or 1.2 6-4 PPs per 17 kb telomere. 6-4 PPs were removed

at similar rates in bulk genomic DNA compared to telomeric DNA, and were removed

more rapidly than CPDs (Fig. 10B). About 20% of the 6-4 PPs remained in both genomic

and telomeric DNA by 3 hours and only ~6% remained by 6 hours post UVC exposure.

Hybridization with telomeric and Alu repeat specific probes confirmed equal loading of

telomeric DNA and genomic DNA, respectively, for all time points (Fig. 10A). The

difference between the genomic CPDs and telomeric CPDs formed at 0 hour is

statistically significant (**, p = 0.0031) by two-tailed heteroscedastic Student’s T test.

Page 133

117

161. Sorrentino, J.A., H.K. Sanoff, and N.E. Sharpless, Defining the toxicology of
aging. Trends Mol Med, 2014. 20(7): p. 375-84.

162. Chen, Y., et al., A shared docking motif in TRF1 and TRF2 used for differential
recruitment of telomeric proteins. Science, 2008. 319(5866): p. 1092-6.

163. Su, Y., et al., Multiple DNA binding domains mediate the function of the ERCC1-
XPF protein in nucleotide excision repair. J Biol Chem, 2012. 287(26): p. 21846-
55.

164. Ahmad, A., et al., Mislocalization of XPF-ERCC1 nuclease contributes to reduced
DNA repair in XP-F patients. PLoS Genet, 2010. 6(3): p. e1000871.

165. Opresko, P.L., et al., Telomere-binding protein TRF2 binds to and stimulates the
Werner and Bloom syndrome helicases. J Biol Chem, 2002. 277(43): p. 41110-9.

166. Wu, Y., T.R. Mitchell, and X.D. Zhu, Human XPF controls TRF2 and telomere
length maintenance through distinctive mechanisms. Mech Ageing Dev, 2008.
129(10): p. 602-10.

Similer Documents