Chapter 10 Flashcards
Mismatch repair removes errors that escape
Proofreading. Mismatch repair system challenges:
A. It must scan the genome for mismatches, and rapidly find and repair the mismatches;
B. It must correct the mismatches accurately. It must replace the misincorporated nucleotide in the newly synthesized strand and not the ___.
E. Coli mismatch repair pathway MutS: scan DNA, recognize mismatches from ___. MutS and mismatched DNA recruit __, which in turn activates __, an endonuclease enzyme that causes an incision or a nick on one strand near the site of the mismatch.
___unwinds the DNA, and the exonuclease progressively digests the displaced single strand, extending beyond the site of mismatched nucleotide. DNA Pol III fills the gap, and DNA ligase seals the nick.
Mismatch repair removes errors that escape
Proofreading. Mismatch repair system challenges:
A.It must scan the genome for mismatches,
and rapidly find and repair the
mismatches;
B.It must correct the mismatches accurately. It must replace the misincorporated nucleotide in the newly synthesized strand and not the correct nucleotide in the parental strand.
E. Coli mismatch repair pathway MutS: scan DNA, recognize mismatches from the distortion they cause in the DNA backbone. MutS and mismatched DNA recruit MutL, which in turn activate MutH, an endonuclease enzyme that causes an incision or a nick on one strand near the site of the mismatch.
Specific helicase (UvrD) unwinds the DNA, and exonuclease progressively digests the displaced single strand, extending beyond the site of mismatched nucleotide.
Dam Methylation at replication fork:
E. Coli Dam methylase methylates ___ on both strands of the sequence 5’-GATC-3 ’.
(a)Replication generates ___; (Methylation of DNA is much slower than DNA synthesis)
(b)MutH then makes ___. Methylation is therefore a “memory” device that enable the E. Coli repair system to retrieve the correct sequence from the parental strand if an error has been made during replication
Dam Methylation at replication fork:
E. Coli Dam methylase methylates A residues on both strands of the sequence 5’-GATC-3 ’.
(a)Replication generates hemimethylated DNA; (Methylation of DNA is much slower than DNA synthesis)
(b)MutH makes incision in unmethylated daughter strand. Methylation is therefore a “memory” device that enable the E. Coli repair system to retrieve the correct sequence from the parental strand if an error has been made during replication
Eukaryotic mismatch repair:
•Eukaryotic cells also repair mismatches and do so using homologue to MutS (MSH: mutShomologs) and MutL(MLS and PMS).
•Eukaryotes have multiple MutS-like proteins with different specificity: one for mismatch, others for small insertion and deletion.
•___(___) mutation leads to genetic predisposition to colon cancer.
•Eukarytotic cells lack __and the clever trick of using ___ to tag the parental strand. They take advantage of the nick after lagging-strand synthesis. How about the leading strand?
• __interacts with sliding clamp component of the
___, and would thereby be recruited to the site of discontinuous DNA synthesis on the lagging strand. Interaction with the sliding clamp could also recruit mismatch repair protein to the 3’ end of the leading strand
Eukaryotic mismatch repair:
•Eukaryotic cells also repair mismatches and do so using homologue to MutS (MSH: mutShomologs) and MutL(MLS and PMS).
•Eukaryotes have multiple MutS-like proteins with different specificity: one for mismatch, others for small insertion and deletion.
•MutHhomolog (MSH2) mutation leads to genetic predisposition to colon cancer.
•Eukarytotic cells lack MutH and the clever trick of using hemimethylation to tag the parental strand. They take advantage of the nick after lagging-strand synthesis. How about the leading strand?
• MSH interacts with sliding clamp component of the
Replisome PNCA, and would thereby be recruited to the site of discontinuous DNA synthesis on the lagging strand. Interaction with the sliding clamp could also recruit mismatch repair protein to the 3’ end of the leading strand
Common types of hydrolytic damage:
(A)____creates uracil, which will preferentially pair with adenine. Adenine and guanine are also subjected to spontaneous deamination, converting them to __ (pair to C) and ___(pair to C but with only two hydrogen bonds).
(B) Depurination of guanine by hydrolysis creates___
.(C) Deaminationof ___ generates a natural base in DNA thymine. (DNA methylation is the result of __, and is involved in transcriptional ___. Thank god 5’mC is not a natural base. But methylated Cs are indeed ___ for spontaneous mutations in vertebrate DNA)
Common types of hydrolytic damage:
(A) Deamination of cytosine creates uracil, which will preferentially pair with adenine. Adenine and guanine are also subjected to spontaneous deamination, converting them to hypoxanthine (pair to C) and xanthine (pair to C but with only two hydrogen bonds).
(B)Depurinationof guanine by hydrolysis creates apurinicdeoxyribose
.(C)Deaminationof 5’-methycytosine generates a natural base in DNA thymine. (DNA methylation is the result of methyltransferase, and is involved in transcriptional silencing. Thank god 5’mC is not a natural base. But methylated Cs are indeed hot spots for spontaneous mutations in vertebrate DNA)
Damage Caused by Alkylation of Bases
•Alkylation is a process where electrophiles: ___(3 steps)
–Encounter negative centers (on the bases or phosphates)
–Attack them
–Add carbon-containing groups (alkyl groups: methyl or ethylgroup)
Damage Caused by Alkylation of Bases:
Alkylating agents like ethylmethane sulfonate (EMS) __.
–Some alkylations don’t change base-pairing, innocuous
–Others cause DNA replication to stall
•Cytotoxic
•Lead to mutations if ___.
–The third type changes ___ properties of a base, so they are mutagenic
Alkylating agents like ethylmethane sulfonate (EMS) add alkyl groups to bases
–Some alkylation don’t change base-pairing, innocuous
–Others cause DNA replication to stall
•Cytotoxic
•Lead to mutations if cell attempts to replicate without damage repair
–Third type change base-pairing properties of a base, so are mutagenic
Damage Caused by Radiation:
•Ultraviolet rays: a wavelength of __ nm is strongly absorbed by the bases.
–Comparatively low energy
–Result in formation of pyrimidine dimers, also called __
pyrimidine dimers (CPDs)
•Gamma and x-rays: used therapeutically kill rapidly proliferating cells in Cancer treatment.
–Much more energetic
–___molecules around the DNA, cause double strand DNA breaks
–Form highly reactive ___ that attack DNA, alter bases, break strands
Damage Caused by Radiation
•Ultraviolet rays: a wavelength of ~260 nm is strongly absorbed by the
bases.
–Comparatively low energy
–Result in formation of pyrimidine dimers, also called cyclobutane
pyrimidine dimers (CPDs)
•Gamma and x-rays: used therapeutically kill rapidly proliferating cells in Caner treatment.
–Much more energetic
–Ionize molecules around the DNA, cause double strand DNA breaks
–Form highly reactive free radicals that attack DNA
•Alter bases
•Break strands
Ex. of Radiation mutation:
Thymidine dimer Cyclobutane ring formation between adjacent thymines by ultraviolet light. This linked bases are incapable of __ and cause ___ to stop during replication.
Thymidine dimer Cyclobutane ring formation between adjacent thymines by ultraviolet light. This linked bases are incapable of base pairing and cause DNA Polymerase to stop during replication.
Direct reversal of DNA damage:
UV radiation damage to DNA can be directly repaired by a ___, which is actually two separate enzymes that catalyze repair of CPDs (cyclobutane
pyrimidine dimer).
Uses energy from near-UV to blue light to break bonds holding ___ together
Direct reversal of DNA damage:
UV radiation damage to DNA can be directly repaired by a DNA photolyase, which is actually two separate enzymes that catalyze repair of CPDs (cyclobutane
pyrimidine dimer).
Uses energy from near-UV to blue light to break bonds holding 2 pyrimidines together
Damage Repair: Methyl group removal:
O6 alkylations on guanine residues can be directly reversed by the “suicide enzyme, ___. This enzyme accepts the alkyl group onto the sulfur group of one of its cysteines and becomes____.
Damage Repair: Methyl group removal
O6 alkylations on guanine residues can be directly reversed by the “suicide enzyme, O6-methylguanine methyltransferase. This enzyme accepts the alkyl group onto the sulfur group of one of its cysteines and becomes irreversibly inactivated
Excision Repair
Excision repair: the damaged DNA is removed, and replaced with the fresh one. Two mechanisms:
Base excision repair and nucleotide excision repair.
•Base excision repair (BER): acts on subtle base damage. Begins with ___(explain). DNA repair enzymes: Remove the remaining deoxyribose phosphate. Replace it with a normal nucleotide
•Nucleotide excision repair: ___
- Base excision repair (BER): acts on subtle base damage. Begins with DNA glycosylaseextrudes a base in a damaged base pair. Clips out the damaged base. Leaves an apurinicor apyrimidinicsite (AP) that attracts DNA repair enzymes•DNA repair enzymes: Remove the remaining deoxyribose phosphate. Replace it with a normal nucleotide
- Nucleotide excision repair: generally deals with more drastic changes to the bases, many of them distort DNA double helix
oxoG:A repair= a fail-safe system
Oxidation of guanine produces oxoG. The modified base can be repaired before replication by __ . If replication occurs before oxoG is removed, resulting in the misincorporationof an A, then a fail-safe __
can remove the A, and allows it to be replaced by a C. This provides a second opportunity for the DNA glycosylase to remove the modified base.
Another example of a fail-safe system is a glycosylase that removes ___ (assume T is from 5-methylcytosine and selectively removes the T so that it can be replaced with a C.)
Oxidation of guanine produces oxoG. The modified base can be repaired before replication by DNA glycosylasevia base excision pathway.If replication occurs before oxoG is removed, resulting in the misincorporationof an A, then a fail-safe glycosylase
can remove the A, and allowing it to be replaced by a C. This provides a second opportunity for the DNA glycosylase to remove the modified base. Another example of a fail-safe system is a glycosylase that removes a T opposite a G. (assume T is from 5-
methylcytosine and selectively removes the T so that it can be replaced with a C.)
Point mutations:___
•Mutation hot spots: certain sites of the chromosome with high frequency mutation rate. Overall rate of normal mutation: __ to __
•DNA microsatellites: Mutation-prone sequence with repeats of ___. Ex., Stretches of CA repeats are found at many widely scattered sites in the chromosomes of human and other eukaryotes. The replication machinery has___, frequently undergoing “slippage”. This slippage increases or reduces the number of ___. As a result, the CA repeat length at a particular site on the chromosome is often highly polymorphic in the population. This polymorphism provides a convenient physical marker for mapping ___.
Point mutations: mutations that alters a single nucleotide
•Mutation hot spots: certain sites of the chromosome with high frequency mutation rate. Overall rate of normal mutation: 10~-6 to 10~-10.
DNA microsatellites: Mutation-prone sequence with repeats of di-, tri-, or tetranucleotide sequences. Ex., Stretches of CA repeats are found at many widely scattered sites in the chromosomes of human and other eukaryotes. The replication machinery has difficulty copying such repeats accurately, frequently undergoing “slippage”. This slippage increases or reduces the number of copies of the repeated sequence. As a result, the CA repeat length at a particular site on the chromosome is often highly polymorphic in the population. This polymorphism provides a convenient physical marker for mapping inherited mutations in certain diseases
Transitions: __to __or __to __substitutions, such as T to C and A to G.
Transversions: __to __or __to __substitution, such as T to G or A, or A to C or T.
Other mutations: Deletion or insertion of one or
more nucleotides
Transitions: pyrimidine to pyrimidine or purine to purine
substitutions, such as T to C and Ato G.
Transversions: pyrimidine to purine or purine to pyrimidine substitution, such as T to G or A,or A to C or T.
Other mutations: Deletion or insertion of one or
more nucleotides
