Mismatched Repair System
Steps (Methylation...)
- Correction of rare mismatches left after replication in E.coli improves fidelity of replication by factor of 10^2-10^3.
- The mismatches are nearly always corrected to reflect the information in the old (template) strand.
- So the repair system must somehow discriminate against the template and newly synthesised strand.
- The cell does this by tagging the template DNA with methy group to distinguish it from newly synthesised strands.
- Dam methylase → methylates DNA, in specific all of the adenines within (5') GATC sequences. (in the parent strand and therefore distinguishes the parent strand from the daughter strand). The daughter strand remain unmethylated after replication so can be distinguished.
- Immediately after passage of replication fork, there is a short period during which template strand is methylated but newly synthesised (daughter) strand is not.
- Therefore the cell is able to recognise the parent and the daughter strands and distinguish between them (due to the methylation of adenine within GATC)
- So if there are any errors in the daughter strands, they can be found (by comparing it to the parent strand!)
Further Steps (Mut Proteins, UvrD, DNA Polymerase, DNA ligase...)
- MutS → binds to a mismatched base pair (wrong nucleotide incoporated!),
- MutS will recruit MutL, as although it can recognise mismatched base pair and bind to it, it can't distinguish between a parent (template) and newly synthesised (daughter) strand.
- MutL → this will bind to the methylated region (adenine on GATC!) and therefore is able to distinguish between the parent and daughter strand.
- MutL protein forms a complex with with MutS protein.
- MutS2-MutL2 complex, bring the mismatched area closer so that it can be cleaved (loops out DNA!)
- MutH recruited by MutL, MutH protein binds to MutL and GATC sequences encountered by MutL-MutS complex.
- MutH endonuclease binds and excises mismatched bases.
- MutH has a site-specific endonuclease activity.
- MutH nicks the daughter strand near the hemimethylated site and recruits a UvrD helicase (DNA Helicase II) to separate the two strands with a specific 3' to 5' polarity
- Another protein called UvrD incorporated, function as helicase (so can separate the strand, where nicks/cuts have been made by MutH).
- A gap is formed, where UvrD along with MutH have cleaved off a section.
- DNA polymerase I recruited to fill the gaps (resyntheises fragment)
- Any other nicks in the strand will be sealed of by DNA ligase.
References
For more detailed info go --> Page 994-996, Lehninger, Principles of Biochemistry, 5ed
https://www.youtube.com/watch?v=41AJ1CFSqKA --> good video!
For more detailed info go --> Page 994-996, Lehninger, Principles of Biochemistry, 5ed
https://www.youtube.com/watch?v=41AJ1CFSqKA --> good video!