Chapter 9 Flashcards
If a germ line of a species contained a high mutation rate, what could be the consequences? What about for the soma?
High mutation rate in the germ line would destroy the species
High mutation rate in the soma would destroy the individual
What are two important sources of mutations?
- inaccuracy in DNA replication (caused by tautomerization)
two consequences:
1. permanent change to DNA coding sequences of genes or its reg sequences
2. chemical alteration to DNA preventing its use as a template for replication and transcription - chemical damage to genetic material
- class of insertions generated by DNA elements (Transposons)
What is a two fold challenge for the cell in terms of mutations?
- it must scan the genome to detect errors in synthesis and damage to the DNA
- it must mend the lesions and do so in a way that, if possible, restores the original DNA sequence.
What are the two types of simple mutations of just one base pair for another?
Transitions - pyrimidine to pyrimidine / purine to purine
Transversions - pyrimidine to purine / viseversa
What is a point mutation?
A simple mutation of an insertion or deletion of a single nucleotide
What is the overall rate at which new mutations arise spontaneously at any given site on the chromosome?
10^-6 to 10^-11 per round of DNA replication.
-Some sites on the chromosome being “hot spots” where mutation arise at a high frequency and other sites undergoing alterations at a comparatively low frequency.
What is the normal mechanism which corrects wrongly incorporated nucleotides?
3’-5’ exonuclease component of the replisome
What happens if a base is misincorporated during replication, and not fixed prior to the second round of replication?
The mutation become permanently incorporated in the DNA sequence
what are the nucleotide mismatch combinations that will go undetected by proofreading exonuclease mechanism?
Three nucleotides on the daughter strand T:G:C, all can be paired opposite of T on the template strand.
T:G:C, T:C:G, G:T:C, G:C:T, C:T:G, C:G:T etc….
T:T:T T:T:T T:T:T T:T:T T:T:T T:T:T
The same variations mirrored will be another six, therefore there are a total of 12 possible combinations which will go undetected. Once the chromosome undergoes its second round of replication, the misplaced (C,G,T:T) combination(s) will be complimentary sequenced and will become permanently encoded as a mutation.
What is the second line of defense for mismatched sequences which go undetected by exonuclease of the replisome? How does it affect replication accuracy?
Mismatch repair system,
increases accuracy by 200-300 orders of magnitude
What challenges does the MMR face?
two challenges:
1 -It must scan the genome for mismatches, which can become transient if not detected prior to second round of replication.
2 -It must correct the mismatch accurately; by replacing misincorporated nucleotide in the newly synthesized strand and not the correct nucleotide in the parental strand.
What are mismatches detected by in E. Coli?
Dimer of MMR called MutS:
scans the DNA, recognizes mismatches from distortions in backbone. MutS embraces mismatch, inducing kink and conformational change to MutS. DNA kink MutS complex together recruits MutL, who then activates MutH to incise or nick on one strand near the site of the mismatch. Then helicase UvrD (1 of 3), unwinds DNA starting from incision moving in direction of mismatch site, and the exonuclease progressively digests the displaced single strand, extending to and beyond the site of the mismatched nucleotide. This process induces a “single-strand gap”, which is then filled in by DNA Pol III and sealed w/ DNA ligase.
How does E. coli mismatch repair system know which of the two mismatched nucleotides to replace?
E. coli tags the parental strand by transient hemimethylation via enzyme “Dam Methylase”
What is Dam Methylase?
E. coli enzyme which methylates both strands of the sequence 5’-GATC-3’, which passes through the replication fork resulting in daughter DNA duplexes being hemimethylated (methylated only on the parental strand)– In this way, the parent strand is always marked immediately after replication or until Dam methylase catches up and methylates the newly synthesized strand.
How is an E. Coli newly synthesized strand marked?
It lacks a methyl group (which is how it can be recognized as the strand for repair, in the event)
What is MutH, what does it require and where does it go?
MutH is part of the Mismatch Repair System. It is a protein which is recruited by the MutS & MutL complex, in which MutH will bind to a nearby Hemimethylated site (5’-GATC-3’), and nick the daughter strand.
If MutH If cleaved DNA on the 5’ side of the mismatch, which exonuclease will degrade that strand and it what direction?
Exonuclease VII or RecJ
Degrades DNA in a 5’ to 3’ direction, removes the stretch of DNA from the MutH-induced cut through the misincorporated nucleotide.
If MutH If cleaved DNA on the 3’ side of the mismatch, which exonuclease will degrade that strand and it what direction?
Exonuclease I
degrades DNA in a 3’ to 5’ direction. After which DNA Pol III fills in the missing sequence.
In eukaryotic cells, what is it’s equivalent to e. coli’s MutS and MutL respectively?
MSH homologs for MutS
MLH & PMS for MutL
Do eukaryotic cells have a homolog of MutH as seen in E. Coli?
No
prior to ligation of okazaki fragments, these fragments are already separated from previously synthesized DNA by a nick - can be thought of as the equivalent of e. coli’s nick created by MutH on newly sythesized strand.
What is the most frequent and important kind of hydrolytic damage to DNA?
Deamination of the base Cytosine.
-under normal physiological conditions, cytosine undergoes spontaneous deamination, thereby generating the unnatural base “Uracil” in DNA, which isn’t supposed to be in DNA.
After deamination of cytosine, if uncaught, and upon the second round of Replication, what will be the resultant mutation in the daughter strand?
Since Cytosine deaminates into Uracil, and Uracil preferentially bonds with Adenine, the mutation would be an Adenine in place of what should have been a Guanine to pair with Cytosine
If adenine spontaneously deaminates, what does it become and what is the resultant mutation?
Adenine spontaneously deaminates into hypoxanthine, which will bind to cytosine in the resultant daughter strand
If guanine spontaneously deaminates, what does it become and what is the resultant mutation?
xanthine, which will bind to cytosine in the resultant daughter strand.
