Exam 3 DNA repair mechanisms

DNA repair system

maintains integrity of genetic material

counteract genetic damage that would result in genetic diseases and cancer

proofreading and mistmatch repair

DNA polymerase “proofreads”, removes and replaces incorrectly inserted nucleotides

mismatch repair becomes activated if proofreading fails

mismatches are detected, cut, and removed, correct nucleotide inserted by DNA polymerase

exonucleases

cut off the ends of a sequence

endonucleases

cut out mismatches within a sequence

strand discrimination

based on DNA methylation

adenine methylase (enzyme in bacteria) recognizes DNA sequences and adds methyl group to adenine residues

newly synthesized strand of replication remains unmethylated

mismatch repair recognizes unmethylated strand and repairs

DNA polymerase

“proofreads”, removes and replaces incorrectly inserted nucleotides

has a 3’ to 5’ proofreading activity which repairs errors induced by incorrect base incorporation during replication

has exonuclease activity: lowers the rates of errors 100 fold to 10^-7 , now 1 mistake in every 10,000,000 bases then 100,000

post replication repair

responds after damaged DNA has escaped repair and has failed complete replication

RecA protein directs recombination exchange with corresponding region on undamaged parental strand (donor DNA)

gap can be filled in by repair synthesis

E.coli SOS repair system

last resort to minimize DNA damage

DNA synthesis becomes error-prone; inserts random/incorrect nucleotides in places that normally would stall replication

bacteria induce expression of 20 genes; their products allow replication to move forth

SOS repair can itself become mutagenic; allows cells to survive with DNA damage (cell would otherwise kill itself)

photoreactivation repair (PRE)

seen in E.coli

photoreactivation enzyme cleaves bonds between thymine dimers, reversing effect of UV radiation on DNA

enzyme must absorb photon of light from blue end of the visible light spectrum to cleave dimer

humans and other organisms lack this repair

base and nucleotide excision repair

light-independent DNA repair mechanisms exist in all prokaryotes and eukaryotes and involve excision repair

endonuclease recognizes and cuts distortion/error

DNA polymerase inserts complementary nucleotides in missing gap

DNA ligase seals final “nick”

base excision repair (BER)

corrects DNA containing a damaged DNA base

DNA glycosylase recognizes altered base

different glycosylases for each type of nucleotide and have ability to recognize nucleotides that have had their structure altered by a tautomeric shift, deamination, alkylation or other modification

when recognized, an altered base they attach to it and rotate the nucleotide breaking the hydrogen bond that holds it to the base on the complementary strand, nitrogenous base is then removed from sugar backbone molecule

nucleotide excision repair (NER)

repairs bulky lesions that alter/distort double helix

recognizes mispaired bases or dimers as they cause a distortion in the shape of double helix

purine + purine: too wide or pyrimidine + pyrimidine: too narrow

remove around 20 bases on either side of the incorrect base

DNA polymerase and ligase will come in and add nucleotides and seal “nick'“

Xeroderma Pigmentosum (XP)

rare genetic disorder due to defects in nucleotide excision repair (NER) pathways: inability to repair damaged caused by UV radiation, often have freckles on their skin

affected individuals exhibit severe skin abnormalities, skin cancers, developmental and neurological defects

individuals have 2000-fold higher rate of cancer

heterokaryon cells

formed by fusing two cells together to form one in cell culture, one with ability of NER and one not capable (XP cell)

if cell is unable to carry out unscheduled DNA synthesis (normal) the two cells have deficiencies in the same gene

if the cell can carry out unscheduled DNA synthesis when fused, but not when they are individual cells it means that one cell has one deficient gene and the other cell has a different deficient gene

when together the two genes complement one another

double-strand break repair (DSB repair)

extremely dangerous

results in chromosomal rearrangements, cancer, cell death

this repair reattaches strands: two pathways → homologous recombination repair or non homologous end joining

homologous recombination repair pathway

recognizes break, digests 5’ end (done by exonucleases), and leaves 3’ overhang

3’ end aligns with sequence complementary on sister chromatid

sister chromatid is unwound and two strand separate, 3’ ends of the damaged strand base pair with sister chromatid using it as template strand

they extend outward past the point where the break occurred, damaged strands then separate from sister chromatid and base pair with one another

any remaining gaps are filled in by DNA polymerase and DNA ligase while the sister chromatids return to its double stranded configuration

occurs during late S or early G2 phase of cell cycle

nonhomologous end joining

activated in G1 prior to replication

repairs double-strand breaks

complex of proteins is involved in end joining

may include kinase and BRCA1 (involved in breast cancer)

proteins bind free ends and ligate ends back together

transposable elements (transposons)

“jumping genes” that can move within and between chromosomes

insert themselves into various locations within genome

found in all organisms; precise function is still unknown

structural features of transposable elements

contain an open reading frame (ORF) that encodes enzymes transposase

may also contain ORFs encoding other proteins in addition to transposase

also contain inverted terminal repeats (ITRs) are short DNA sequences that are inverted relative to each other

direct repeats (DRs) flank the DNA transposon in the chromosomal DNA

Steps in DNA transposon transposition

1. transposase cleaves DNA at ITRs

2. Transposase makes staggered cuts at target site in chromosomal DNA

3. transposase inserted transposon at target site

4. gaps in target site are filled in by DNA polymerase and DNA ligase

Insertion sequences (IS elements)

move from one location to another

cause mutations if inserted into gene/gene-regulatory region

Bacterial transposons

larger than IS elements

can introduce multiple drug resistance to bacterial plasmids; move from plasmids to bacterial chromosomes, spreading multiple drug resistance between strains

Ac-Ds system in Maize

mobile genetic elements in corn plants found using this system

dissociation (Ds) and activator (Ac) are two mutations of transposable elements in corn, mobile controlling elements (transposable elements)

movement of Ds gene is dependent on Ac gene

Barbara McClintock

nobel prize physiology or medicine 1983

found some kernels are white, some are blue and some are speckled because of transposable elements

Effects of Transposable Elements

if a TE inserted into a protein coding region, it can disrupt the protein (null mutation)

if a TE inserts itself into a promoter, it can prevent a gene from being expressed

if a TE that is inserted into a promoter contains a promoter for one of its own genes, it can cause a gene to be expressed when it usually isn’t (or shouldn’t)

types of transposable elements in plants

autonomous elements: can transpose (jump) by themselves, are self contained

nonautonomous: cannot transpose by themselves and require the assistance of an autonomous element to provide the necessary gene products

McClintock’s discovery

if a corn plant carries the C gene it produces anthocyanin which makes the kernels purple

some mutants called c mutants are colorless because the anthocyanin is not deposited in the cells

in some cases there is a reversion during development and pigment is deposited in certain cells giving a speckled appearance

if reversion occurs early in development there is more purple tissue in the kernels

hypothesized that the c mutation was occuring when a “mobile controlling element” called Ds for dissociation was inserted into the C gene

Ds requires the help of an activator Ac to insert itself

the Ac activator can also excise the Ds element causing the reversion

Ac and Ds

Ac is an autonomous transposable element: can copy itself, move to a new location and insert itself; can produce the enzyme transposase

Ds is a non autonomous transposable element; it cannot copy itself or move to a new location by itself; requires the assistance of Ac which codes for the enzymes that provide the machinery to copy, move and insert it into a new location; it does not produce transposase and uses the enzyme produced by Ac

Mechanism of Ds/Ac

retrotransposons

transposable elements that amplify and move within the genome; use RNA as an intermediate

two types: the long terminal repeat (LTR) retrotransposons or the non-LTR retrotransposons

both can be either autonomous or nonautonomous

How retrotransposons work

1. RNA polymerase present in the cell transcribes the retrotransposon

2. the RNA transcript is translated by the cell’s ribosomes and produces two proteins, reverse transcriptase and integrase

3. the reverse transcriptase transforms the RNA transcript of the retrotransposon into double stranded DNA

4. integrase cuts a section of DNA and inserts the DNA version of the retrotransposon into a new location

retrotransposon structure

LTRs contain open reading frames that encode the enzymes integrase and reverse transcriptase (RT)

transcription promoters and polyadenylation sites are located within the 5 and 3 long terminal repeats (LTRs)

non-LTR retrotransposons also encode integrase and reverse transcriptase but are lacking LTRs: transcription promoters and polyadenylation sites are located within 5 and 3 untranslated regions (UTRs)

Steps in retrotransposon transposition

1 +2: Retrotransposons is transcribed and translated by cellular enzymes

3. reverse transcriptase creates double-stranded DNA copies of each retrotransposon RNA

4. integrase inserted double-stranded DNA copies into chromosomal DNA

Copia Elements in Drosophila

more than 30 families of TEs found

copia: class of DNA element in Drosophila transcribed into “copious” amounts of RNA

dispersed throughout genome and transposable to different chromosomal locations

(DTR- direct terminal repeat (267 bp long and ITR- inverted terminal repeat 17bp)

Effects of Copia Elements in Drosophila

a. white gene in wild-type drosophila contains 6 exons, all of which are present in the mRNA

b. the white gene in mutant white-apricot drosophila contains an insertion of copia (red) in the second intron and a prematurely terminated mRNA containing only 2 exons

c. revertant drosophila have lost most of the copia element form the second intron, resulting in wild-type mRNA and wild-type eye color

hybrid dysgenesis

mutation due to high rates of P element transposition in germ line: elevated mutation rates and sterility caused by TEs

P element in drosophila

discovered during study of hybrid dysgenesis

use as tools for genetic analysis: vectors

crosses between lab stocks from the early 1900s and wild caught flies produced extremely high rates of sterility and mutations (hybrid dysgenesis) only occured when wild caught (new) males were crossed to lab stock females from old stocks; reciprocal crosses were not affected

very useful for producing transgenic drosophila: can inject plasmids with P elements and a specific gene into an egg and it will insert the gene into the genome of the host cell’s germ line

P (new wt strains) and M (old wt) strains

P strains have the P element M strains do not

P strain females produce a protein that suppresses the transposition of the P element, M strain females do not

M strain egg cytoplasm has no protection from transposition leadings to hybrid dysgenesis

P strain eggs have suppressor protein so they are unaffected (also M strain sperm does not contain P elements)

Cross of male/female with or without P element

P male x M Female: sterile because P elements disrupt the genome in eggs

P female x M male: viable because P females produce a suppressor protein that prevents P element transpositions and M males do not have P elements

human transposable elements

half of human genome is composed of TEs

LINEs and SINEs: long interspersed elements and short interspersed elements

0.2% of detectable human mutations may be due to transposable element insertions: transposons may contribute to variability that underlies evolution