BIMM 100: Week 2

end replication problem

when final RNA primer at the extreme 5' end of the daughter lagging strand is removed, DNA polymerase cannot fill the gap

solution to end replication problem

extend the 3’ end of the PARENT strand

telomerase

a reverse transcriptase enzyme that contains its own template RNA

telomerase RNA component

contains nucleotides that are complementary to the 3’ end of the parental DNA strand, continuously translocates to create repeats

telomerase protein component

the actual enzyme that performs reverse transcription

The character of the telomerase, NOT the character of the chromosome….

determines the telomere sequence

function of telomeres

protection from nucleases and regulation of cell replicative capacity

3-20kb of repeated sequence

indefinite replication

reduced number of repeated sequences

limited replication

no repeated sequences

replication stops, cell death

55kb of repeated sequences

cancer

endonuclease

cleave bonds WITHIN a sequence

exonuclease

cleaves bonds at the ENDS of a sequence (free 3’-OH or 5’-PO4)

Additional feature of DNA polymerase

3’ to 5’ exonuclease proofreading activity (“backtracking” direction), removes MOST RECENTLY misincorporated nucleotide

mismatch excision repair

incorrect base in daughter strand is recognized, mutated DNA is removed by nucleases, gap is repaired by DNA polymerase and DNA ligase

How do endonucleases know which is the daughter strand?

Daughter strand is UNMETHYLATED

oxygen radicals/alkylating agents

chemical nucleotide conversion

UV light

thymine dimers (covalently bonded thymines in same strand)

DNA damaging drugs

double stranded DNA breaks

base deamination

cytosine turns into uracil, or 5-methyl cytosine turns into thymine. requires base excision repair

base excision repair

incorrect base removed from phosphate backbone by glycosylase, APEI endonuclease and AP lyase remove the phosphate, gap filled with correct base by DNA polymerase and DNA ligase

nucleotide excision repair (fixes “bulky” DNA lesions" such as thymine-thymine dimers)

distortion is recognized, helix opens, endonucleases remove damaged DNA strand, gap is filled by DNA polymerase and DNA ligase

Repair by Nonhomologous End Joining (NHEJ) (repairs double stranded breaks)

Recognition of broken ends, exonucleases remove overhanging nucleotides, ligation. OR translocation from different chromosomes. results in an erroneous sequence

repair by homologous recombination

uses an identical sister chromatid as a template to repair damaged dsDNA, error free

Cas9

RNA-guided DNA nuclease, creates double-stranded breaks in DNA to edit genome