DNA Damage and Repair

DNA damage, DNA repair, Cancer

Sources of DNA mutations and damage

spontaneous chemical reactions

DNA replication

Transposons

Mutagens - substances that cause DNA mutations (UV light, ionizing radiation (x-ray and gamma ray), chemical mutagens)

Spontaneous hydrolysis

damages nucleotides; ex. depurination or deamination

Depurination

removing entire purine; similar to deletion mutation

Deamination

removing amino group; similar to substitution mutation

ex. deamination of cytosol results in uracil

Without ___, replication of damaged nucleotides causes mutation

DNA repair;

Deamination → mutated strand with different base pair

Depurination → mutated strand with deleted pair

Transposons and retrotransposons

genes that code for proteins that move them in the genome → can disrupt other genes

__ is cut out and inserted at new location

Retrotransposon

transcription → translation → using reverse transcriptase, reverse transcription of RNA to DNA → synthesis of second DNA strand → new copy of retrotransposon is inserted into

Transposons play important roles in ___ and ___

evolution and brain development

ex. retrotransposition gave rise to differing sperm cells, leading to population variants

Ultraviolet (UV) Light DNA damage

cause adjacent nitrogen bases to bond to each other (called cross-linking)

introduces variety of mutations during DNA replication; not a suitable template and cannot be transcribed correctly

Chemical mutagens

cause mutations by attaching to DNA ex. deletions or insertions

Ionizing radiation

causes double strand breaks (DSBs)

DSBs can lead to ___

translocations

What happens during translocation?

piece of DNA moves in genome from one place to another

ex. gene from one chromosome moves to another chromosome, producing a mutated daughter cell

Mechanisms for repairing damaged DNA 

direct reversal

single strand damage repair

double strand break repair

Direct reversal

reversing what was changed (what error happened);

photoreactivation (undoing dimers caused by UV by breaking cov. bond that formed)

methyl group (CH3) removal

Single strand damage repair

base excision repair (BER)

nucleotide excision repair (NER)

mismatch repair (MMR)

Double strand break repair

non-homologous end joining (NHEJ)

microhomology-mediated end joining (MMEJ)

homologous recombination**

Base excision repair

1. Uracil DNA glycosylase removes uracil from DNA → DNA helix with missing base (abasic site)

2. AP endonuclease (removes abasic site from backbone) and phosphodiesterase remove sugar phosphate

3. DNA polymerase adds new (correct) nucleotide

4. DNA ligase seals nick

How does the DNA machinery know which nucleotide in a deaminated C mutation was wrong?

G was the correct nucleotide in the mismatched pair because U is not used in DNA

Nucleotide excision repair

1. excision nuclease cuts out part of strand with damaged DNA (dimer caused by UV or ionizing radiation)

2. DNA helicase unwinds strands to separate out segment

3. DNA polymerase + ligase repairs area with correct (non-dimerized) nucleotides

Two mechanisms for DSB repairs

nonhomologous end joining

homologous recombination

nonhomologous end joining

joining 2 open ends of DNA;

1. processing of DNA ends - removing some nucleotides via deletion (frame shift)

2. end joining

3. result - deletion of some DNA sequence

What are the problems with nonhomologous end joining?

no way to know the 2 pieces were originally with each other

creates deletion mutation

homologous recombination

requires same DNA sequences; uses sister chromatids as template to repair across break

steps of homologous recombination

1. nuclease digests/trims 5’ ends of both broken strands

2. strand exchange by complementary base pairing → one strand invades sister chromatid strands

3. repair polymerase synthesizes DNA using undamaged DNA from sister chromatid as template

4. invading strand is released from sister chromatid → goes back to find original strand pair to base pair with it

5. broken double helix reforms

6. DNA synthesis continues using strands from damaged DNA as template

7. DNA ligase connects gaps

8. double strand break is accurately repaired

When in the cell cycle is homologous recombination possible?

during S, G2, and M phases

What is crucial for cancer prevention?

genomic stability - cells should only divide if genome is stable

Mutations in what type of genes lead to cancer?

cell cycle control genes

Tumor suppressors

genes that code for proteins that increase genomic stability; suppresses cell division

Oncogenes

overstimulates/promotes cell cycle and cell division

p53

tumor suppressor; present in all cells but usually inactive and ubiquitylated

active in damaged DNA to regain genomic stability; pauses cell cycle or leads to apoptosis

is mutated in >50% of human cancers

p53 is a ___ for p21

transcription factor

p21

inhibits G1 and S CDKs; is a CDK inhibitor protein

Myc

oncogene that when overexpressed, activates p53

Cancer develops (quickly/gradually)

gradually

Multistep model of cancer development

alterations in several cell cycle control genes accumulate → as you age, more mutations develop (hence increased chance of cancer)

DNA repair enzymes role in cancer

fix mutations, helps slow process of accumulating alterations in cell cycle control genes

most cancers have malfunctioning ___ mechanisms

BRCA 1 and BRCA 2

tumor suppressors; proteins that recognize mutated/damaged DNA and initiate DNA repair

BRCA 1 and 2 mutations are also risk factors for ___

ovarian cancers and other cancers

Loss of ___ leads to cancer

heterozygosity; easily inherited BRCA mutations

How does loss of heterozygosity lead to cancer?

inherited BRCA mutation + BRCA mutation that occurs naturally in a somatic cell → zero functional copies of BRCA-1; 2 broken copies

Cancer cells show a lot of ___ in their genome

irregularities; ex. wrong number of chromosomes, wrong pairs of chromosomes, etc.

more mutations

Genetic instability and cancer cells

cancer cells just damaged enough to be a problem but not damaged enough to die; cells with too much genetic instability cannot survive → use of radiation to kill cancer cells because it makes them too unstable to survive