Genetics Exam 2 - Telomeres+Recombination+Repair+CRISPR

Why chromosomes would shorten

RNA Primers removed from ends of chromosomes cannot be replaced by polymerase I (needs an existing 3’-OH to build off of).

Telomere

Terminal structure of eukaryotic cells that insure maintenance/replication of ends of linear chromosomes. Repeating units (TTAGGG)

Telomerase

RNA+protein, reverse transcriptase:synthesizes DNA from RNA template. Telomerase’s RNA portion is a primer matching DNA telomeres, telomerae reverse transcribes its own RNA to the opposite DNA strand’s end, primase and polymerase build the complementary strand. Always leaves a small protion of ssDNA, but elongates telomeres by mutliple repeats increasing protection.

Telomeres and Disease

Cancer cells have high telomerase activity, aging is attributed to shortening of telomeres (Werner syndrome - premature aging due to loss of function telomerase).

Homologous Recombination

Formation of heteroduplex where homologous DNA cross over (one strand from one chromosome pairs with its complement on another).

Proof that recombination occurs after DNA replication

Not all haploids are recombinant (2 of 4 chromatids recombine, 2 stay the same → diversity in gametes).

Recombination Mechansim

Single strand or double strand break → exhcnage → rejoin, heteroduplex.

Recombination Steps

1. Enzyme breaks phosphodiester bond of both strands on one chromatid

2. 5’ end of each broken strand degraded by exonuclease → 3’ ssDNA tails

3. ss tail invades non-sister chromatid → heteroduplex formed, displacement loop stabilized by ss binding porteins

4. Double holliday junction formed when D loop and other ss tail form heteroduplex, DNA synthesis replaces and reseals breaks/gaps

5. Heteroduplex region lengthend as wave migrates

6. Resolution via resolvase and ligase

Noncrossover recombinants

Vertical cut resolution → arms did not swap when resolved, only small portion of DNA altered, alleles likely did not recombine (any change was due to repair).

Crossover recombinants

Horizontal cut resolution → arms did swap when resolved, alleles likely recombined.

NHEJ

Double stranded break in DNA, cell rejoins ends of DNA but there could be mistakes due to lack of original template.

HDR

Homology directed rejoining → recombination allows for proper repair of double stranded break using non-sister chromatid template.

CRISPR Cas-9

Cas-9 protein uses guide RNA matching region of DNA to target and cleave specific target if adjacent to protospacer adjacent motif. Approved to treat sickle cell.

Types of CRISPR Mutations

NHEJ: prone to frameshifts→knockouts

HDR: Uses template matching non cleaved regions to add specific sequence

Double NHEJ: Cleave out a larger portion of DNA.