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

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

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

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

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

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

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

Surveillance For all types of DNA damage 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

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

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

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

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

Thymine Dimer - a common DNA lesion

Nucleotide excision repair - Part I (bacteria)

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

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

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.

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 A A Examples: C T G T 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?

Determination of new strand (bacteria) A T CH 3 DNA replication New strand A T CH 3 A T New strand + CH 3 Re-methylation (slow) A T CH 3 A T + CTAG CH 3 GATC CH 3

Mismatch repair (bacteria) A T CH 3 G T DNA replication CH 3 Mismatch Recognition mutS G T CH 3 Binding of mismatch factors mutH mutL G T CH 3 G T Translocation of mutL and mutH to hemimethylated site CH 3

G T Mismatch repair (bacteria) A T CH 3 DNA synthesis by DNA Pol, SSB & ligation T Exonuclease digestion CH 3 Nicking of non-methylated (new) strand by mutH G T CH 3 Nick A T CH 3 Re-methylation

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

Reduction in error rate Base pairing can lead to error frequency of ~ (i.e., errors per nucleotide incorporated) DNA polymerase actions (polymerase specificity and 3’ --> 5’ proofreading) can lead to error frequency of ~ 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

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

AT is a genetic disorder with a incidence of 1 per 40,000 births Ataxia Telangiectasia (AT) 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

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

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

p53 status is a determinant of tumor response to therapy p53 +/+p53 -/- + adriamycin ) Lowe, Science 266:807, 1994 tumor volume (cm 3 ) + adriamycin

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

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