DNA replication and repair - Lecture 3

1. DNA replication and repair - Lecture 3 Jim Borowiec September 28, 2006

2. Overview of DNA replication Telomere Centromere Telomere DNA chromosome Origin of DNA replication Specialized elements termed 'origins of DNA replication’ occur many times on a chromosome Initiation of DNA replication from origin of replication generates structures termed 'DNA replication bubbles'

3. 3’ 3’ 5’ 5’ DNA replication (from internal regions of the chromosome) by leading strand synthesis 3’ + 5’ by lagging strand synthesis RNA primer iDNA Processing loss of DNA DNA 3’ 3’ 5’ replication 5’ End replication problem After multiple rounds of DNA replication, genetic information will be lost

4. (TTAGGG)nTTAGGGTTAGGG-3’ (TTAGGG)nTTAGGGTTAGGGTTAGGG-3’ (AATCCC)nAATCCC-5’ (AATCCC)nAATCCC CCAAUCCC Telomerase 5’ RNA template CCAAUCCC 5’ • Telomerase adds one or more copies of the TTAGGG repeat, preventing DNA loss Replication of telomeres • Telomeres contain many copies of a specific DNA repeat • Involves special RNA-containing polymerase called telomerase

5. Germ line cells Germ line cells Germ line cells p53 mutation p53 mutation ‘Hayflick limit’ Somatic cells Somatic cells Somatic cells Telomerase activation Telomere length Telomere stabilization ‘Hayflick limit’ ‘Hayflick limit’ Senescence Senescence Crisis Crisis Widespread cell death Widespread cell death Senescence Cell Divisions Telomerase needed for cell immortalization • Most somatic cells do not have telomerase activity

6. Mechanisms to repair damaged DNA or mispaired DNA • Usually involves synthesis of portions of only one DNA strand • Involves synthesis of 1 to >1000 nt depending on type of repair reaction

7. Types of DNA damage 1. Spontaneous A. Base deamination (ex: cytosine is converted to uracil at a rate of ~100 bases per human cell per day) B. Loss of bases - depurination and depyrimidation (~5000 purines are lost per human cell per day) C. Oxidative damage to bases (life span of an organism is inversely correlated with metabolic rate/DNA oxidation) 2. Environmental damage A. Radiation (ionizing and ultraviolet) B. Chemical agents (e.g., benzo[a]pyrene)

8. Surveillance factors with different recognition specificities continually scanning the DNA for damage or mispairs • Upon finding a damage or mistakes, surveillance factors recruit other repair factors Signal to recruit additional repair factors Surveillance • For all types of DNA damage

9. N H O 2 + N H 4 H N H H N H H O N O H O N 2 sugar-phosphate sugar-phosphate backbone backbone Cytidine Uridine Deamination of cytidine to uridine (spontaneous)

10. N H 2 + N H 4 H N H O N H O 2 Recognized by DNA glycosylase Uridine in DNA repaired by Base Excision Repair (BER) O H H N H O N sugar-phosphate sugar-phosphate backbone backbone Cytidine Uridine

11. MANY DNA GLYCOSYLASES EXIST DIFFER IN SUBSTRATE SPECIFICITY GENERALLY RECOGNIZE MONO-ADDUCT DAMAGE

12. N H 2 + N H 4 H N O H O N H O H 2 H N H O N sugar-phosphate sugar-phosphate backbone backbone Cytidine Uridine H O 2 Uracil-DNA glycosylase O H O + H sugar-phosphate H N Free uracil backbone H O N AP site H Uridine in DNA repaired by Base Excision Repair (BER)

18. Thymine Dimer - a common DNA lesion

19. Nucleotide excision repair - Part I (bacteria)

20. Nucleotide excision repair - Part II (bacteria) (uvrD)

21. Human nucleotide excision repair (NER) • Xeroderma pigmentosum (XP) - an inherited disease in which patients show an extreme sensitivity to sunlight • XP is a result of mutation of various genes involved in NER

22. Xeroderma Pigmentosum Society, Inc. Camp Sundown for XP children The program schedule is 9:00 p.m. to 5:00 a.m. to maximize night time hours for play and minimize need for protective arrangements.

23. Examples: A C G A T T Mismatch repair Primary source of DNA alterations arising during normal DNA metabolism is mispairing of bases during DNA synthesis In eukaryotes, deamination of 5-methyl cytosine generates a thymine (and a T:G base pair) and is corrected by mismatch repair Question: Bases are not damaged, only incorrectly paired. How does the mismatch repair machinery determine which is the correct base and which is the incorrect base?

24. CH3 CH3 GATC A CTAG T CH3 CH3 CH3 CH3 CH3 DNA replication CH3 A T New strand + A T New strand Re-methylation (slow) CH3 + CH3 A A T T Determination of new strand (bacteria)

25. DNA replication CH3 G A T T CH3 CH3 CH3 CH3 CH3 Mismatch Recognition mutS G T mutH Binding of mismatch factors mutL Translocation of mutL and mutH to hemimethylated site G G T T Mismatch repair (bacteria)

26. Nick Nicking of non-methylated (new) strand by mutH G G T T CH3 CH3 CH3 CH3 CH3 Exonuclease digestion T DNA synthesis by DNA Pol, SSB & ligation Re-methylation CH3 A A T T Mismatch repair (bacteria)

27. Hereditary nonpolyposis colon cancer (HNPCC) • HNPCC is a hereditary cancer syndrome with individuals having increased incidence of colon cancer, ovarian cancer, and endometrial tumors • Caused by defects in human mismatch repair genes that are homologous to bacterial mismatch repair genes - Defects in hMSH2 (human mutS homolog) account for ~60% of HNPCC cases - Defects in hMLH1 (human mutL homolog) account for ~30% of HNPCC cases • Cells from HNPCC patients are 100-fold more mutable than normal patients

28. Reduction in error rate Base pairing can lead to error frequency of ~10-1 -10-2 (i.e., errors per nucleotide incorporated) DNA polymerase actions (polymerase specificity and 3’ --> 5’ proofreading) can lead to error frequency of ~10-5-10-6 Accessory proteins (e.g., SSBs) can lead to error frequency of ~10-7 Post replicative mismatch repair can lead to error frequency of ~10-10

29. G2/M (ATM, p53) Ionizing radiation S Severe damage S phase (ATM, ATR, ...) G1/S (ATM, p53) Apoptosis Involvement of ATM and p53 in the cellular checkpoint response M G2 G1

30. Ataxia Telangiectasia (AT) AT is a genetic disorder with a incidence of 1 per 40,000 births Approx. 10% of individuals with AT develop neoplasms, such as Hodgkin’s disease, with most of these occurring with people less than 20 years of age The overall cancer incidence in homozygotes is ~100-fold increased. AT individuals have defects in gene encoding the checkpoint kinase ATM

31. The tumor suppressor p53 • The 'guardian of the genome’ • The most frequently mutated gene in cancer • Functions as a sequence-specific transcription factor regulating a large number of genes • Responsive to a wide array of signals that stress the cell including: • DNA damage • hypoxia • hyperproliferative signals emanating from oncogenes

32. p53-dependent apoptosis suppresses tumor growth Choroid plexus epithelium Van Dyke, 1994

33. p53 status is a determinant of tumor response to therapy p53 +/+ p53 -/- + adriamycin tumor volume (cm3) + adriamycin ) Lowe, Science 266:807, 1994

34. Non-critical gene or non-coding sequence Little or no effect on cell viability Non-repaired mismatch Essential region of essential gene Cell death through apoptosis Gene involved in growth stimulation or tumor suppression Potential for unregulated cell growth Additional mutations (genomic instability) Cancer Pathway of Carcinogenesis DNA replication

35. (tumor suppressor) (proto- oncogene) (proto- oncogene) (tumor suppressor) (tumor suppressor) p53 APC PTGS2 Ras 18q LOH Mutation of: Early Adenoma Late Adenoma Normal cell Carcinoma Pathway of Carcinogenesis (colorectal cells)