DNA Replication and Repair Disorders Disorder Frequency Defect Fanconi’S Anaemia 1/22,000 in Some Deficient Excision Popns

THE EUKARYOTIC CELL CYCLE

S phase DNA synthesis G1 DNA replication Growth phase 1 Main checkpoints and repair

G2 Go M Growth phase 2 Quiescent Mitosis

1 REPLICATION START REPLICATION – GENERAL PRINCIPLES Must be ready

Must all start at the same time Must know where to start ACCURACY/FIDELITY

Proof reading/repair FINISH Must distinguish between original and copy

Must all finish Must ensure that each piece of DNA is replicated only once Therefore must know where to finish

2 REPLICATION START REPLICATION – GENERAL PRINCIPLES Must be ready : G1

Must all start at the same time G1  S Must know where to start ACCURACY/FIDELITY ORIGIN OF REPLICATION Proof reading/repair G2 FINISH Must distinguish between original and copy

EPIGENETICS

Must all finish complete S Must ensure that each piece of DNA is replicated only once Therefore must know where to finish

3 DNA polymerase DNA Polymerase - Matches the correct nucleotides then joins adjacent nucleotides to each other Provides an RNA primer to start polymerization

Unwinds the DNA Figure 5-4 Molecular Biology of the Cell (© Garland Science 2008) and melts it Keep the DNA single stranded DNA synthesis always occurs by adding after it has nucleotides to the 3’-OH of the growing strand. been melted by helicase Synthesis is always in the 5’- 3’ direction

4 Replication Enzymes • Helicase - Unwinds the DNA and melts it • Single Strand Binding Proteins - Keep • DNA Polymerase - Matches the correct the DNA single stranded after it has been nucleotides then joins adjacent melted by helicase nucleotides to each other • Gyrase - A topoisomerase that Relieves • Primase - Provides an RNA primer to torsional strain in the DNA molecule start polymerization • Ligase - Joins adjacent DNA strands • Telomerase - Finishes off the ends of together (fixes “nicks”) DNA strands

5 Causes of DNA Damage •Chemical mutagens •Radiation •Free radicals

Figure 5-41 Molecular Biology of the Cell (© Garland Science 2008)

6 RADIATION = ENERGY RADIOLYSIS OF WATER

H O . OH +. H + e- 2 ENERGY DEPOSITION IN DNA S-A…….T-S P P S-A…….T-S S-A…….T-S .OH P P P S-A…….T-S A-S P P P P S-T…… A-S C-S P P S P S-G……C-S P G - S P P S - G P S-C….....G-S P S S-C P P .OH P S-T……..A-S P P P S-T S-A……..T-S P P P S-A……..T-S DNA DAMAGE P P

7 RESPIRATION AND AGEING

Carbohydrate + O2

Energy + CO2 + H2O

.0 H O . OH 2 2 2

DNA damage ~10000 lesions/cell/day Maynard et al, 2008

8 Types of DNA Damage: Base Loss and Base Modification Adenine tautomer

Chemical Modification Depurination Photodamage thymine dimer

Deamination Chemical Modification by O2 free radicals

9 Maintenance of DNA Sequences DNA Polymerase as Self Correcting Enzyme Generation of a • Correct nucleotide has greater affinity for mutation by the moving polymerase than incorrect nucleotide adenine • Exonucleolytic proofreading of DNA polymerase tautomer – DNA molecules with mismatched 3’ OH end are not effective templates; polymerase cannot extend when 3’ OH is not base - About every 1 paired 4 in 10 bases – DNA polymerase has separate catalytic site that removes unpaired residues at terminus

10 Maintenance of DNA Sequences DNA Polymerase as Self Correcting Enzyme Two catalytic sites

11 DNA damage repair pathways. DNA repair mechanisms in cells

• Base-excision repair (PARP) • Nucleotide-excision repair • Recombinational repair (BRCA1/BRCA2) • Mismatch repair

12 DNA damage repair pathways. DNA Repair Base Excision Repair a. DNA glycosylase recognizes damaged base b. Removes base leaving deoxyribose sugar c. AP endonuclease cuts phosphodiester backbone d. DNA polymerase replaces missing nucleotide e. DNA ligase seals nick

13 Strand Directed Mismatch Repair System Poly (ADP-ribose) polymerase (PARP) • Removes replication errors not recognized by • A key regulator of DNA damage repair processes replication machine • Involved in DNA base-excision repair (BER) • Detects distortion in DNA helix • Binds directly to DNA damage • Methylation occurs shortly after replication occurs • Produces large branched chains of poly (ADP-ribose) • Reduces error rate 100X • Attracts and assists BER repair effectors • 3 Step Process recognition of mismatch excision of segment of DNA containing mismatch resynthesis of excised fragment

XRCC1 Lig3 PNK Polß

Tutt A on behalf of ICEBERG investigators. ASCO 2009;Abstract CRA501

14 Strand Directed Mismatch Repair in Mammals

• Newly synthesized strand is preferentially nicked and can be distinguish in this manner from parental strand • Defective copy of mismatch repair gene predisposed to cancer

15 DNA Repair

DNA Repair ►Despite 1000’s of alterations that occur in DNA each day, few are retained as mutations DNA Damage Can Activate Expression of ►Efficient repair mechanisms Whole Sets of Genes ►Importance of DNA repair highlighted by: • Heat Shock Response • SOS Response Number of genes devoted to DNA repair

 mutation rates with inactivation or loss of DNA repair gene ►Defects in DNA repair associated with several disease states

16 Failure of DNA repair

• When DNA repair fails, fewer mutations corrected  increase in number of mutations in the genome.

• The protein p53 monitors repair of damaged DNA. • If damage too severe, p53 protein promotes programmed cell death (apoptosis)

• Mutations in genes encoding DNA repair proteins can be inherited  overall increase in mutations as errors or damage to DNA no longer repaired efficiently.

17 Protein dynamics to and from sites of DNA breaks. DNA replication and repair disorders Disorder Frequency Defect Fanconi’s anaemia 1/22,000 in some Deficient excision popns. repair Hereditary nonpolyposis 1/200 Deficient mismatch colon cance repair Werner’s syndrome 3/1,000,000 Deficient helicase Xeroderma pigmentosum 1/250,000 Deficient excision repair

18 OMIM 278700) XP xeroderma pigmentosum

Caused by homozygosity For a recessive mutation in A repair gene.

One example of a DNA-repair genetic disease