Chapter 14 lecture (repair systems) Flashcards
two types of nucleotide excision repair
global genome repair
transcription-coupled repair
three types of excision repair
nucleotide
base
mismatch
nucleotide excision repair
recognizes bulky lesions in DNA
global genome repair
repairs damage everywhere in the genome
transcription-coupled repair
repairs damage in the transcribed strand of active genes
base excision repair
recognizes damage to individual nitrogenous bases
mismatch excision repair
A type of repair that corrects mispaired bases
Usually immediately following replication
Preferentially corrects the sequence of the daughter strand
Distinguishes between the daughter strand and parental strand
Sometimes based on methylation status
Also recognizes insertion and deletion hairpin loops
single base changes
Affect the DNA sequence but don’t greatly distort the structure
May occur due to in situ mutations or replication errors
Mismatch persists only until the next replication
Limited time for repair
structural distortions
Provide a physical impediment to replication or transcription
Pyrimidine dimers
Alkylation
Nitrogenous base removal
repair system recognition
single base changes
structural distortions
short patch repair
The Uvr system makes incisions approximately 12 bases apart on both sides of damaged DNA
99% of repair
DNA between the incisions is then removed
Excision
New DNA is synthesized to fill the gap
long patch repair
Remaining 1%
1500-9000 nucleotides removed and repaired
Decision mechanism of short- versus long-patch are unknown
Base excision repair is triggered by direct removal of
a damaged nitrogenous base from DNA
The enzymes that remove bases from DNA are called
glycosylases
Glycosylases sometimes also have …. activity that cleaves the ring structure of the deoxyribose sugar
lyase
glycosylase action alone
leads to the DNA Pol gamma epsilon pathway that results in the replacement of a long stretch of nucleotides
Long-patch pathway
both glycosylase and lyase action
leads to the DNA Pol beta pathway that results in the replacement of a short stretch of nucleotides
Short-patch pathway
Damaged DNA that has not been repaired causes
DNA Polymerase III to stall during replication
DNA polymerase V or DNA polymerase IV can temporarily
replace polymerase III and synthesize a complement to the damaged strand
Error-prone polymerases add
random nucleotides to daughter strand at site of damage
error prone synthesis
The DNA synthesized by repair DNA polymerases often have errors in their sequence
Methylation and directionality of mismatch
The strand lacking methylation at a Me-A/T is usually replaced in prokaryotes
Repair must be completed before daughter strand is methylated
Methylation status is not used by eukaryotic repair enzymes
Nuclease enzymes that resect 5’ ends of broken strands become very active in
S and G2
When sister chromatids are present
In G1 phase,
no sister chromatids are present, so recombination repair would use homologous chromosome for repair
Loss of heterozygosity
CRISPR
A Cas9 transgene can be transferred into a cell along with a guide RNA that targets a specific DNA sequence in the genome and the gene
Cas9 and guide RNA are usually included together on an expression vector
The Cas9/guide RNA complex binds to the target sequence in the genome and creates a DbSB
DbSB is repaired using recombination repair mechanism
If homologous sequence is present, it will replace the damaged region
Gene editing
Homologous sequence is sometimes introduced into the cell on same vector as Cas9 and guide RNA
If the homologous recombination DbSB repair mechanisms cannot find an identical or homologous sequence to use as a template for repair, then the cell will use the
nonhomologous end-joining (NHEJ) pathway
nonhomologous end-joining (NHEJ) pathway
Primary pathway used to repair DsDB in mammalian somatic cells
Spend most of their time in G1 when identical or homologous sequence is less available
The NHEJ pathway is error-prone and usually results in
errors in the repaired DNA
the NHEJ pathway is acceptable in what cells
Acceptable in somatic cells because most of the mammalian genome is not essential for life and can tolerate NHEJ errors
NHEJ mechanism
Ku complex binds to ends and bridges them
Initial ends are not completely blunt
Some nucleotides are lost from each strand after breakage
Leads to small, irregular non-complimentary single stranded overhangs
Initial bridging is facilitated by tiny stretches of short (as little as 1 bp) complimentary sequences
Insertions and deletions commonly result from
NHEJ
Different patterns of histone modification may distinguish different
repair pathways or stages of repair
H2AX phosphorylation is a conserved double-strand break-dependent modification that
Recruits chromatin modifying enzymes
Facilitates assembly of repair factors
Remodelers and chaperones are required to reset chromatin structure after
completion of repair
