Lecture 8

DNA repair and transposons and a little bit on retroviruses

Mismatch repair (MMR)

A type of repair that corrects mispaired bases, typically immediately following replication

• The process preferentially corrects the sequence of the daughter strand by distinguishing the daughter strand and parental strand, sometimes on the basis of their states of methylation.

• Done by DNA Pol III the main replicase for both DNA strands (my addition)

The human genome has many repair genes

• Direct reversal of damage: numerous genes

• Base excision repair: 15 genes

• Nucleotide excision repair: 28 genes

• Mismatch excision repair: 11 genes

• Recombination repair: 14 genes

• Nonhomologous end-joining: 5 genes

• DNA polymerase catalytic subunits: 16 genes

Photoreactivation

A repair mechanism that uses a white light-dependent enzyme to split cyclobutane pyrimidine dimers formed by ultraviolet light.

Excision repair

a type of repair system in which one strand of DNA is directly excised and then replaced by resynthesis using the complementary strand as template.

Base excision repair (BER)

A pathway of excision repair that recognizes damage to single bases, such as deamintation or alkylation, and either repairs the base alone (short-patch repair) or replaces 2-10 nucleotides (long-patch repair).

Nucleotide excision repair (NER)

An excision repair pathway that recognizes bulky lesions in DNA (such as UV-induced pyrimidine dimers).

• NER is divided into two major subpathways:

  • Transcription-coupled repair (TC-NER), which repairs damage in the transcribed strand of active genes

  • Global genome repair (GG-NER), which repairs damage anywhere in the genome

Repair systems recognize DNA sequences that do not conform to

standard base pairs

Excision systems

remove one strand of DNA at the site of damage and then replace it

Deamination is reversed by replacing

Uracil with Cytosine

• Since deamination is the mutation of cytosine into uracil

Replication errors introduce mismatched base pairs

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Methylation can distort the structure of DNA

Guanine gets mutated into methyl-guanine which causes distortion in the double helix which us corrected by dealkylation.

Depurination requires base replacement

Adenine mutates and depurniates which causes a purine to be missing in the double helix corrected by insertion.

Thymine dimers must be removed by excision

Thymine mutation by UV irradiation into a thymine dimer which causes distortions in the duplex and is repaired by excision.

Recomibnation-repair systems use

recombination to replace the double-stranded region that has been damaged.

All of these repair systems are prone to

introducing errors during the repair process.

Photoreactivation is

a non mutagenic repair system that acts specifically on pyrimidine dimers

Excision repair systems in E.Coli

The Uvr system:

• Damage, a mutant mbase is mismatched and/or distorts structure which is recognized

• Incision ~12 bases (short patch) apart on both sides of damaged DNA. Endonuclease cleaves on both sides of damaged base

• Excision: exonucleases or helicase removes DNA between the nicks

• Synthesis: Polymerase synthesizes replacement DNA

• Sealing of nicks with ligase

Transcribed genes are

preferentially repaired when DNA damage occurs

Xeroderma pigmentosum (XP)

is a human disease caused by mutation in any one of several nucleotide excision repair genes.

• Numerous proteins, including XP products and the transcription factor TF₁₁H, are involved in eukaryotic nucleotide excision repair.

Eukaryotic Nucleotide Excision Repair Pathways

Global genome repair and Transcription-coupled repair

Global genome repair recognizes

damage anywhere in the genome

Transcriptionally active genes are

preferentially repaired via transcription-coupled repair

Global genome repair and transcription-coupled repair differnt in their mechanisms of damage recognition by

using XPC and RNA Polymerase II respectively.

Base excision repair systems require glycosylases

Uracil and alkylated bases are recognized by glycosylases and removed directly from DNA

Base excision repair is triggered by directly removing a damaged base from DNA

Base removal triggers the removal and replacement of a stretch of polynucleotides

The nature of the base removal reaction determines which of two pathways for excision repair is activated.

The polδ/ε pathway replaces a long polynucleotide stretch

The polβ pathway replaces a short stretch of DNA

Glycosylases and photolyase (a lyase) act by

flipping the base out of the double helix , where depending on the reaction, is either removed or modified and returned to the helix.

When Uracil is removed from DNA

glycosylases remove the base from the sugar phosphate backbone

Deamination of unmethylated cytosine produces

Uracil

It is due to deamination that DNA is most likely the genetic material

Since it is more stable

Deamination of methyl cytosine produces

thymine

base removal triggers

excision repair

• the long patch pathway

• or the short patch pathway

Damaged DNA that has not been repaired causes DNA polymerase III to stall during replication

DNA polymerase V (coded by umuCD) or DNA polymerase IV (coded by dinB) can synthesize a complement to the damaged strand

The DNA synthesized by repair DNA polymerases often has errors in its sequence and is thus called

error-prone synthesis

Mutator

A mutation or a. mutated gene that increases the basal level of mutation

• Such genes often code for proteins that are involved in repairing damaged DNA

The mut genes code for

a mismatch repair system that deal with mismatched base pairs

There is a bias in the selection of which strand to replace at mismatches

It is the strand that is lacking methylation at a hemimethylated GATC/CTAG site

The mismatch repair system is used to remove errors in a newly synthesized strand of DNA.

At G-T mismatches, the T is

preferentially removed

Replication slippage generates

a single strand loop

MutS/MutL repair replication slippages

A replication slippage generates a single strand loop and then MutS binds to the mismatch and then MutL binds which allows for the mismatch to be removed by exonuclease, helicase, DNA polymerase, and ligase

The rec genes of E.coli code for

the principal recombination-repair system

The recombination-repair system functions

when replication leaves a gap in a newly synthesized strand that is opposite a damaged sequence

Single strand exchange

when the single strand of another duplex is used to replace the gap of a damaged strand

Recombination-repair uses two duplexes

Damage: Bases on one strand of DNA are damaged →

Replication generates a copy with gap opposite damage and a normal copy. →

Retrieval:

Gap is repaired by retrieving sequence from normal copy →

Gap in normal copy is repaired

The yeast RAD mutations, identified by radiation-sensitive phenotypes, are in genes that code for repair systems.

THe RAD52 group of genes is required for recombination repair

The MRX (yeast) or MRN (mammals) complex is required to form a single-stranded region at each DNA end.

Nonhomologous End-Joining also repairs double strand breaks

Nonhomologous end-joining (NHEJ) pathway can ligate blunt ends of duplex DNA

Mutations in double-strand break repair pathways cause human diseases

NHE-J requires several reactions

End-recognition → Trimming → Filling → Ligation

Both histone modification and chromatin remodeling are essential for repair of DNA damage in chromatin

DNA damage in chromatin requires chromatin remodeling and histone modification

Different patterns of histone modifications may distinguish stages of repair or different pathways of repair

Remodelers and chaperones are required to reset chromatin structure after completion of repair

RecA triggers the SOS system

Damage to DNA causes RecA to trigger the SOS response, which consists of genes coding for many repair enzymes

RecA activates the autocleavage activity of Lexa

LexA represses the SOS system; it autocleavage activates those genes

LexA and RecA have a reciprocally antagonistic relationship.

Transposon (transposable element)

A DNA sequence able to insert itself (or a copy of itself) at a new location in the genome without having any sequence relationship with the target locus.

Retrovirus

An RNA virus with the ability to convert its sequence into DNA by reverse transcription

A major cause of sequence change within a genome is the movement of a transposon to a new site

Transposon generates new copy at random site → unequal crossing over occurs between related sequences

Transposons are mobile genetic elements fundamental to our own biology and genetic engineering

Retrotransposon (retroposon)

a transposon that mobilizes via an RNA form; the DNA element is transcribed into RNA, and then reverse-transcribed into DNA, which is inserted at a new site in the genome. IT does not have an infective (viral) form.

• commonly, retrotransposons containing long terminal repeats (LTSs) are referred to as retrotransposons, while non-LTR containing retrotransposons are referred to as retroposons

Insertion sequences are simple transposition modules

An insertion sequence is a transposon that codes for the enzyme(s) needed for transposition flanked by short inverted terminal repeats

The target site at which a transposon is inserted is duplicated during the insertion process

• two repeats in direct orientation at the ends of the transposon

The length of the direct repeat is:

• 5 to 9 bp

• characteristic for any particular

Inverted terminal repeats

flank the transposon, they read the same way in each direction as you approach the transposon

Transposase

is the enzyme which recognizes the inverted repeats and is responsible for transposition

Transposons can

carry other genes in addition to those coding for transposition

Composite transposons have

a central region flanked by an IS element at each end

Either one or both of the IS elements of a composite transposon may

be able to undertake transposition

A composite transposon may transpose as a unit

An active IS element at either end may also transpose independently

Transposition occurs both

replicative and nonreplicative mechanisms, conservative and non-conservative

IS sequences can mobilize any region of DNA

All transposons use a common mechanisms

• staggered nicks are made in target DNA

• the transposon is joined to the protruding ends

• the gaps are filled

Replicative Transposon

Transposons is copied to a new site:

• Donor remains unaltered

• Recipient gains copy of transposon

Non replicative, non-conservative

Transposons is copied to a new site:

• Donor has a break at site of transposon

• Recipient gains copy of transposon

Conservative transposition

Every bond is preserved,

• Movement conserves bonds in both the donor and recipeint

Homologous recombination between multiple copies of a transposon causes

rearrangement of host DNA

• excision (can delete in between)

• inversion (or invert between)

Excision

Direct repeats recombine to excise material

Pairing of direct repeats → Recombination releases material between repeats as circular molecule

Inversion

Inverted repeat recombination inverts material

• Inverted repeats pair → forms a loop or a holiday junction → inverted region

Homologous and Nonhomologous end joining (NHEJ)

repairs host DNA by cutting and pasting (non-replictive) mechanisms of transposition

Transposition starts by

forming a strand transfer complex. The transposon is connected to the target site through one strand at each end.

Three mechanisms of cut & paste transposition

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Replicative transposition

transposon → donor DNA cleave of transferred strand → strand transfer → replication fork assembly → replication through the transposon → continued replication ligation

Cointegrate with 2 copies

Two types of transposon regulation

• Regulate copy number

• Control target site choice