CMB REVIEW FOR STEP I: Prepared by Pamela L. Derstine, Ph.D. Chromosome Organization Chromosome Organization DNA Replication DNA Replication DNA Repair DNA Repair DNA Recombination DNA Recombination Cell Division Cell Division February 13, 2003

Chromosome Organization

CLINICAL SIGNIFICANCE: 2. Retrotransposon integration into genes inactivation 1. Most unrepaired mutations no effect. 3. Retrotransposon mispairing unequal crossing over deletion or duplication disease. 4. Unequal crossovers of simple sequence repeats bordering chromosome regions microdeletion syndromes.

NF1 Caused by Alu insertion Autosomal dominant 17q11.2 Frequency 1:3000 Also, skeletal deformities Predisposition to tumors of nervous system 50% new mutations

Review Problem #4, August 8, 2001 for data analysis

7q11 Microdeletion: Williams syndrome Autosomal dominant aortic stenosis Unusual face Joint laxity Mental disability Sensitivity to loud noises Strikingly happy personality Remarkable musical & language skills above mental function

DNA Replication

CLINICAL SIGNIFICANCE: Bloom’s Syndrome Helicase deficiency

DNA Replication: telomeres

DNA Replication: telomerase

Clinical significance: Cancer or absent telomere due to progressive shortening with DNA replication

CLINICAL SIGNIFICANCE: CANCER SELF-RENWAL OF EPITHELIAL CELL POPULATION BY REPEATED CELL DIVISION Telomeres shorten and uncap Normal p53 cell cycle checkpoint control Normal senescent cells stop dividing Loss of p53 and cell cycle checkpoint control Mutant cell survives and proliferates Chromosome fusion Chromosome bridge Chromosome breakage Chromosome translocation CHROMOSOME BREAKGE-FUSION- BRIDGE CYCLE Massive chromosomal damage Cell dies due to catastrophic genomic instability and DNA damage Telomerase reactivated Chromosomes are partially stabilized and cell survives with many mutations CANCER

DNA damage, repair mechanisms and consequences

DNA REPAIR DURING REPLICATION: DNA PROOFREADING MISMATCH REPAIR

Proofreading

hMSH2/6 (MutS  ) recognizes mismatches, single-base loops hMSH2/3 (MutS  ) recognizes insertion or deletion loops hMLH1/hPMS2 (MutL  ) binds to MutS and replication factors (determining strand specificity) Excision and resynthesis is carried out by exo and endonucelases, DNA pol  / , RPA, PCNA MMR occurs close to the replication fork

CLINICAL SIGNIFICANCE: HNPCC Autosomal dominant inherited MMR mutation (multiple locations): no polyps LOH Microsatellite instability Additional mutations accumulate Colorectal Cancer: Tumor cells with almost normal karyotype

CLINICAL SIGNIFICANCE: HNPCC Male heterozygotes: 90% lifetime colon cancer risk Female heterozygotes: 70% lifetime colon cancer risk, 40% endometrial cancer risk Other cancers Accumulation of point mutations, microsatellite instability due to strand slippage during replication (replication error positive or RER + phenotype)

RER RER Phenotype Testing (RFLP)

HNPCC vs FAP Familial adenomatous polyposis: autosomal dominant cancer; inherited mutation APC tumor suppressor gene (5q21-q22); early appearance of benign polyps Deletion or mutation of both copies of the tumor suppressor APC begins the process. Each polyp can develop into a carcinoma.

FAP

HNPCC vs FAP 5% population develops colorectal cancer Colorectal cancer: 11% total cancer deaths FAP: 0.5% all colorectal cancers HNPCC: 3.0% all colorectal cancers Sporadic: most colorectal cancers (DS DNA repair defects [translocations], cell division defects [nondisjunction])

DNA REPAIR POST-REPLICATION: EXCISION REPAIR DOUBLE-STRAND BREAK REPAIR

Excision Repair

DNA damage recognition by XPC, XPA, RPA Coupled incisions by XPG (3’) and XPF (5’), generating a nucleotide fragment Helicase subunits of TFIIH (XPB, 3’-5’ and XPD, 5’-3’) unwind and release SS DNA fragment Repair synthesis by DNA polymerase  /  and the accessory proteins PCNA, RPA, ligase.

CLINICAL SIGNIFICANCE: XERODERMA PIGMENTOSUM Heterogeneous group of autosomal recessive skin disorders: sun-sensitivity, dryness and pigmentation of exposed skin, increased tendency to develop tumors Complementation

Double Strand Break Repair DNA damage checkpoint protein ATM activated Phosphorylate p53 to arrest cell cycle Ku, DNA-PK proteins protect ends; mechanistic overlap with V(D)J recombination

CLINICAL SIGNIFICANCE: NHEJ - increased radiosensitivity and immune deficiency when repair proteins inactive HEJ – CANCER-PRONE CLINICAL SYNDROMES

RecBCD homologs generate DS DNA nicks and degrade both strands 5’-to-3’ RecA homologs (RAD51) and accessory proteins (RPA, BRCA1, BRCA2) catalyze strand invasion and loop displacement RuvABC homologs promote branch migration and resolution by strand cutting Repair synthesis by DNA polymerases, PCNA, ligase, etc.

OR SISTER CHROMATIDS (HEJ) BIVALENT IN MEIOSIS Recombination; HEJ

CLINICAL SIGNIFICANCE OF DYSFUNCTIONAL HEJ: 2. Ataxia telangiectasia, an autosomal recessive, pleiotropic, variable disease due to mutations in ATM gene (11q23). 1. Familial breast cancer (BRCA1 [17q21], BRCA2 [13q12.3] mutation, autosomal dominant); one possible mechanism is shown.

Cell Division

Clinical Significance: Cancer drug targets Taxol: binds, stabilizes microtubules Colchicine, colemid: binds subunits, prevents polymerization Vinblastine, vincristine: binds subunits, prevents polymerization Nocodozole: binds subunits, prevents polymerization

Clinical Significance: Cancer drug targets Motor protein inhibitors: example of Monastrol

Clinical Significance: Aneuploidy Trisomy 21: meiotic nondisjunction

Clinical Significance: Sporadic colorectal cancer (mitotic nondisjunction due to checkpoint malfunction)

MAD2 Cdc20 MAD2 Release from kinetochore Clinical significance: metaphase checkpoint malfunction

Clinical Significance: LOH Rb1 gene locus Retinoblastoma (autosomal dominant 13q14); Many common cancers. Review 9/17/01 problem set, #11.