8 - DNA Repair

what must be maintained

genetic stability

most mutations are

accidental

DNA Repair

set of processes that immediately correct spontaneous changes in DNA

what is much DNA resserved for

repair enzyme synthesis

loss of DNA repair gene =

not good

what is the phenotype of Xeroderma pigmentosum (XP)

akin cancer, UV sensitivity, neurological abnormalities

what is the enzyme/process affected in XP

nucleotide excision repair

what are the phenotypes pf cockayne syndrome

UV sensitivity, developmental abnormalities

what is the enzyme/process affected in cockayne syndrome

coupling of nucleotide excision repair to transcription

what is the phenotype in Ataxia telangiectasia (AT)

leukemia, lymphoma, gamma-ray sensitivity, genome instability

what is the enzyme/process affected in AT

ATM protein, a protein kinase activated by ssDNA breaks

what is the phenotype of Brca1

Breast and ovarian cancer

what is the process affected by Brca1

repair by homologous recombination

Depurination

spontaneous loss of ~5000 purine (adenine or guanine) bases lost per cell per day because of hydrolysis of the glycosyl linkage to deoxyribose

what is the most common spontaneous mutation

depurination

what happens during depurination

occurs when the bond connecting a purine to its deoxyribose sugar is broken by a molecule of water, resulting in a purine-free nucleotide that can’t act as a template during DNA replication

what does depurination leave

hydroxyl group on primary carbon instead of base with glycoside bond

what is the result of depurination

results in loss of base pair (deletion) in one daughter cell that inherits this mutation

deamination

spontaneous removal of amine (NH2) group from ~100 cytosines per cell per day to form uracil and an ammonium ion

what does hydrolysis do during deamination

bumps off the NH2 from cytosine which is replaced by an oxygen to form uracil

what does uracil match to

adenine

what is the result of deamination

sequence is changed since uracil wants a A to pair to

what is spontaneous oxidative destruction

reactice oxygen radicals or chemical exposure cause base damage

uv exposure

forms pyrimidine dimers via covalent linkage between two bases

how does pyrimidine dimers work

causes distortion of DNA helix by pulling two thymine bases close together. These block transcription and replication if not repaired

how does non-ionizing radiation (UVA or UVB) cause dimers

by inducing formation of covalent linkages, but a highly efficient system repairs them

is XP heritable

yes , because the mutation happens in the germ line

how is XP ‘s defect expressed

dysfunctional repair enzyme

what is a result of XP

skin cell mutations, resulting in tumors

what does DNA repair depend on

healthy DNA repair genes to make functional DNA repair enzymes/repairs

why is semi-conservative good for DNA repair

one strand still carries the good stuff

what happens if one strand is damaged

complementary one retains intact copy which can be used to restore damaged strand

what are the two repair mechanisms for spontaneous mutations

base-excision repair and nucleotide excision repair

what does BER use to recognize the altered DNA base

glycosylases

describe the 4 BER steps

1. glycosylase (enzyme) scans to find damaged base, once found cuts bond between base and sugar, leaving sugar w/o base

2. AP endonuclease recognize empty site missing base and cuts sugar-phosphate backbone (creates gap in DNA strand)

3. DNA poly uses opposite strand as template and inserts correct nucleotide into gap

4. DNA ligase seals nick w/ new sugar piece

what begins NER

recognition of dimer

steps of NER

1. damage recognition

2. helicase unwinds the DNA around the damage

3. excision nuclease cleaves on both sides of damage leaving a gap of ~30 nucs

4. DNA poly + ligase fill in nucleotides and seal

How is alteration recognized in base excision

Glycosylase enzyme “flips out” each nucleotide so it can be checked by the endonuclease and repaired if necessary

What do the alterations for NER produce

a structural “bulk lesion” in the DNA helix that is atypical

How is a NER alteration fixed

Large multi enzyme complex sees this distortion in the helix and cleaves the phosphodiester backbone of the DNA strand on both sides, then helicase along with DNA pol and ligase deal with the sis containing the lesion

What is DNA repair closely coupled with

Transcription

How is RNA pol part of the repair process

It stalls at DNA alterations (specifically NER) during transcription and calls forth coupling proteins with direct excision repair machinery to these sites

What happens when RNA pol detectes something that must be repaired

RNA pol reverses itself, repair machinery comes in, damage is repaired, and RNA pol restarts

Why is transcription-couples repair the most important

Since it is the DNA that will be transcribed and translated

Where is there a defect in cockayne syndrome

Transcription-coupled repair

What is there an accumulation of in cockayne syndrome

Arrested transcription complexes at lesion sites; RNA pol depleted, transcription of certain genes is insufficient

What are the effects of cockayne syndrome

Underdeveloped tissues and organs; lifespan into 3rd decade

Affect neurons, fibroblasts, and lymphocytes

Unclear as to how children appear normal for ~3 years then disease progresses

What are most pols in Family B

Replication (alpha delta epsilon)

What are most pols in family X

Repairs (beta mu lambda)

What are pols in family Y

Translesion

Characteristics of Translesion polymerases

Contain many that are not too great

Don’t have any exonucleolytic activity

Don’t discriminate about which nucleotide to add

Who comes to the rescue of Family B members during replication

Translesion polymerases (Family Y)

When do double-stranded DNA breaks happen more frequently

Ionizing radiation issues, replication errors, oxidizing agent destruction

What are the methods of repair for dsDNA breaks

• non-homologous end joining

• Homologous recombination

What does nonhomologous en joining include

Removing a set of nucleotides, then lighting the strands back together

What does NHEJ result in

Deletion mutation

Why is the deletion mutation made by NHEJ acceptable

So much of genome is non-gene coding

What pols are involved with NHEJ type of repair

Lambda and mu

Where is NHEJ important

In VDJ joining in lymphocytes

What are bad effects of NHEJ

Can join ends of two different chromosomes together (translocation)

Leads to nucleotide deletion

What is the more accurate repair mechanism for dsDNA

Homologous recombination

When does homologous recombination happen

Only after replication (S and G2 phases) when nearby daughter DNA duplex can serve as template for repair

Negative consequences of homologous recombination

Loss of heterozygosity

Sequestration of repair proteins is controlled by additional proteins such as Brca1 and Brca2; alterations in these cause cancer