WEek 4 Flashcards
DNA cst change
good at keepon info because if one strand is damaged, there’s another one
2000-10000 hydrolytic depurination everyday/cell
1 cytosine deamination everyday/cell
Mutations vs mutagens
Mutations
Permanent
TRansmissible changes to genetic material
Spoontaneous by transposable elements&by error during replication
Mutagens
Chemical compounds (UV radiation/ionizing radiation)
Increase frequency of mutation related to cancer (carcinogen)
Repair of mismatch
DNA polu, 1 error/10000 nucleotide incorp.
Proofreading exonuclease (often part of polym): In Eukaryote, DNA poly, epsilon and omega have a 3’ to 5’ exonuclease but not alpha
VS mismatch repair
mismatch repair
Increases infedily by 1000 fold (1 error/10^9)
Not aprt of the replication fork or replisome
Some P interact with PCNA
Mismatch repair steps
MSH2&SH6 detect mispairing
Triggers binding&activity of MLH1 endocnuclase (dimerized with PMS2)
DNA helicase (not CMG one) unwinds
DNA exonuclease digest segment of daughters strands
Gap filled and repaired by polym omega and DNA ligase
Repair of small modification
BAse excision repair
Dna glycosylase hydrolyzes the ond between mispaired base and sugar P-backbone (provides sepcifity in repair)
Apurinic/Apyrimidiinc endonuclease 1 cuts backbone
AP lyase in polum beta removes deoxyribose phosphate
Polym beta and Dna ligase fill and seal nick in backbone
REpair of bulky lesions
nucleotide excision repair
formation of thymine-hymine dimers that threats the double-helix struc
1-Complex of P23B&XP-C=initial recognition of DNA lesion
2-REcruit transcription faction TFIIH
3-TFIIH catalyzes unwind of DNA region around lesion
4- Process recruits RPA & XP-G (uwinding act)
5- XP-f is recruited… so XP-F & XP-g=endonuclease act (cuts 24-32 naises apart to release fragment/lesion degraded)
6-DNA polum and ligase=fill and lifate ssDNA gap
Double strand break
Mutagen: Ionizing radiation anticacener drugs (eg: bleomycin)
Effect: Single/Double strand breaks=genomic rearrangements and translocations
Repair: Error-prone repair by non-homologous end-joining (NHEJ) or repair by homologous recomb. (HR)
Double strand break expl.
Sing strand breaks=more common4=efficiently repaired by ligase
DNA unwinding during replication could concert a single-stand break into a double one
During meosis, DNA recomb occurs between chromosome from mom&dad to mingle their seq. (Telomer)
homolous recombination (HR)
vs
non-homo end joining (NHEJ)
HR
High fidelity mechanism= repair DNA breaks
Used during S & G2 phase of all cycle
Requires undamaged copy of chromosomes to be repaired
Process requires:
-Processing of breaks to expose ssDNA seq.
-conduct a search for id. seq.
-use them as template of DNA synthesis
-Repair the break
NHEJ
Error-prone (=mutations)
Used during GI phase of cell cycle
complex formed by:
-DNA-dependent P
kinase (DNA-PK)
- Ku 80/Ku 70 heterodimer
binds to the ends of the break
Ends digested by nuclease &ligated tgtr
PCR and DNA sequencing
Fundamental techniques in molecular biology:
Polymerase chain Rx (PCr) and DNA sequencing
Polymerase chain rx use
Used for:
sequencing (some approaches)
DNA cloning (isolating particular gene/overexpression of p)
Detection of pathogens (COVID)
Gene editing (Crispr…)
PCR tubecontent
Tube content:
DNAtemplate
Taq DNA polym
Primers (oligonucleotide/oligos)
dNTPs
Originating from thermophilic
(resist to high T°) Thermus aquaticus
Doesnt have proofreading exonuclease=
mistakes often (use of other enzyme too)
