1. MSH2 and Human Nonpolyposis Colon Cancer Yael Aschner

2. Presentation Outline What is MMR? What is MSH2? What is HNPCC?

3. DNA Mismatch Repair (MMR) Identified in bacteria Mechanism for fixing errors in DNA replication The frequency of mutations introduced by DNA polymerase during DNA replication is 1/1012 base pairs; of these, 99.9% are repaired by MMR.

4. MMR in E. coli Three proteins  MutS  MutL  MutH

5. MMR in E. coli MutS performs first step of MMR: recognition of base/base mismatches and small insertion or deletion loops (IDL) ATPase acting as an asymmetric homodimer Translocates along DNA and promotes DNA loop formation

6. MMR in E. coli MutS undergoes conformation change and interacts with MutL in the presence of ATP MutL stimulates MutH endonuclease activity, which removes newly replicated DNA fragment with mismatch DNA polymerase fills in the gap

7. MMR in E. coli

8. MutS Homolog 2 (MSH2) Human homolog of E. coli MutS Chromosme 2p 15 exons Encodes a 934 amino acid protein Involved in the recognition of mismatched bases and the initiation of the MMR reaction

9. E. coli MutS Crystal Structure The MutS monomer (green) recognizes the G:T mismatch in the DNA (red) and has ADP bound (at the bottom of the figure in red). The monomer in blue provides the non-specific DNA binding domains and an empty ATPase domain

10. Eukaryotic MMR Highly conserved  No MutH homolog Six homologs of MutS (MSH1-MSH6)  Three are needed for initiation of the repair process: MSH2, MSH3, MSH6 MSH2 forms a heteroduplex with MSH6 or MSH3, forming MutS  and MutS , respectively

11. MutS  MutS  can bind to most base-base mismatches and loops of one or a few nucleotides  Present at higher concentrations  Primary mismatch recognition factor  Responsible for most mismatch recognition

12. MutS  MutS  can bind to IDLs with up to ~8 unpaired nucleotides  Not involved in base-base mismatches  Backup system Some overlap between the complexes MSH2 acts as a scaffold while MSH3 and MSH6 provide specific binding

13. Mismatch Recognition

14. MSH2 Inactivation Inability to repair base-base mismatches and small nucleotide insertions and deletions that occur as the result of misalignment or erroneous DNA replication Acts as a TS gene  CANCER

15. Human Nonpolyposis Colon Cancer (HNPCC) Associated with deficiency in MMR Syndrome characterized by familial predisposition to colorectal carcinoma and extracolonic cancers  Gastrointestinal, urological, female reproductive tracts Most common hereditary colorectal cancer syndrome  2%-5% of all colon cancers in Western population Very high penetrance (~85%) Early onset  42 years Autosomal dominant inheritance (cellular level recessive) Treatment: removal of all or most of the colon

16. Colon Adenoma of the Large Intestine Precursor polyps (or adenomas) appears to be no greater than the number found in the general population These polyps are thought to progress more rapidly to cancer and form at earlier ages

17. How is MSH2 Mutated in HNPCC? 40% of HNPCC mutations are in MSH2 gene Predominantly small deletions, insertions, splice site alterations, nonsense or missense mutations MSH2 is the most common gene to have deletions  Range from one exon to whole gene MSH2 or MLH1 mutations responsible for 50%- 80% of HNPCC families 20% of sporadic colorectal cancers due to MMR mutations

18. HNPCC Inheritance Individuals heterozygous for mutations Two Hit Hypothesis Functional wild type allele lost by somatic mutation event  MMR inactivated  Accumulation of mutations

19. Loss of Heterozygosity

20. Mouse Models for HNPCC (Wei, et al) Msh2 +/- mutants indistinguishable from wild type Homozygous Msh2 -/- mice all died by 12 months (50% by 6 months)  Strong predisposition for tumor phenotype  Cell extracts lost ability to repair single-base mispairs and IDLs of up to four nucleotides  Tissues displayed high mutation frequencies Small intestinal tumors, lymphoid tumors