BIO 120.12 | Module 2: DNA Repair Mechanisms

Repairs UV damage (Thymine dimers)

Photoreactivation, nucleotide excision repair

First DNA repair mechanism discovered

Photoreactivation

• First DNA repair mechanism discovered

• UV damage (thymine dimers)

Photoreactivation

• Light-repair enzyme

• Activated by visible light (300 - 600 nm)

• Folic acid (cofactor) absorbs light

• Uses energy from absorbed light to perform cleavage of T-T (thymine) dimers

Photolyase

Enzyme used in photoreactivation to cleave thymine dimers

Photolyase

At what wavelengths is photolyase activated

Visible light (300-600 nm)

Cofactor of photolyase responsible for absorbing light

Folic acid

Explain photoreactivation

1. Exposure to UV causes formation of thymine dimer in dsDNA

2. Photolyase binds to thymine dimer

3. Visible light activates photolyase

   1. Vis light activates folic acid cofactor

   2. Energy from absorbed visible light is used by photolyase to repair thymine dimer

4. Photolyase released, leaving repaired DNA

Allows for localized excision and resynthesis of nucleotides at site of mismatch

Mismatch excision repair

Large structural distortion or chemical modification in the DNA, caused by the attachment of large molecules or chemical groups to the DNA bases

Bulky lesion

Repairs replicative errors within newly synthesized DNA

Mismatch excision repair

Repairs UV-induced damage, bulky lesions, mutations from other causes

Nucleotide excision repair

What type of bond is formed (and cleaved by photolyase) between T-T dimers

Glycosidic bond

T/F: Photolyase is inactive without visible light

TRUE

Methyl-directed post-replication repair system

Mismatch excision repair

T/F: Photolyase only repairs pyrimidine dimers

TRUE

Enzymes that cut DNA at internal sites rather than at ends

Endonuclease

Enzymes that remove nucleotides from the end of DNA, cutting 1 nucleotide at a time

Exonuclease

Explain nucleotide excision repair

1. Recognition of bulky lesion

2. Endonuclease cuts DNA in the middle, exonuclease removes damaged DNA

3. DNA polymerase fills the gap by synthesizing new DNA, using intact complementary strand as template

4. DNA ligase seals remaining gap by joining old & new DNA

• Repairs bulky lesions, base alterations that are results of metabolic factors (e.g., oxygen radicals)

• Damaged or incorrect bases is excised as free bases creating abasic sites, which could be:

  • Apurinic site: lacking a purine base (A,G)

  • Apyrimidinic site: lacking a pyrimidine abse (C,T)

Base excision repair

Explain base excision repair

• DNA glycosylase: Hydrolysis of N-glycosidic bond between incorrect base and sugar in DNA backbone, forming AP site

• AP endonuclease: Removal of AP site, excision of lesion from its 3’ side

• DNA polymerase I: Filling in the resultant gap

• DNA ligase: seals nicked DNA

Activated by stalled replication or major DNA damage

SOS repair system

In BER, why does excision of lesion occur from 3’ side?

AP endonuclease excises AP site from the 3’ end to facilitate the subsequent gap-filling function of DNA polymerase, which always synthesizes new DNA in 5’ to 3’ direction and recognizes 3’ OH, thus adding nucleotides from 3’ end of growing strand.

What happens to the excised bases in base excision repair?

They are released as free bases and can be reused for other anabolic processes.

• Methyl-directed post-replication repair system

• Repairs replicative errors within newly synthesized DNA

• Allows for localized excision and resynthesis of nucleotides at site of mismatch

Mismatch excision repair

An error-prone repair system because it occurs without template

SOS repair system

This is how excision repair enzymes “know” which strand is incorrect if DNA is not physically distorted

Methylase (used for mismatch excision repair)

T/F: Mismatch excision repair is not reflected frequently because they can check if DNA is correct or not.

T/F: Mobile genetic elements are essential to cell

FALSE

Not essential

Mismatch excision repairs often repairs what type of mutation?

Spontaneous mutations

Adds a methyl group to select bases soon after DNA strand is made

Methylase

Segments of DNA with genes encoding their own replication, recombination, transmission

Mobile genetic elements (MGEs)

Who discovered methylase?

Hamilton Smith (1970)

Explain mismatch excision repair

• MMR proteins, such as MutS (MSH), scans and recognizes mismatched base pairs in newly synthesized DNA strand

• Newly synthesized DNA strand is identified by its undermethylated state compared to parental DNA, which is already methylated

• Repair endonuclease (MutH) cuts unmethylated daughter strand near mismatch site

• Exonuclease removes portion of strand including mismatched base

• DNA polymerase fills gap

• DNA ligase seals final nick

• Segments of DNA that have genes encoding their own replication, recombination, transmission rpct

• Not essential to cell

Mobile genetic elements

SOS repair system is activated by _

Stalled replication or major DNA damage

• Activated by stalled replication or major DNA damage

• Allows DNA repair to occur without template; hence, it’s an error-prone repair

• Mutations induced by this are better than cell death

SOS Repair System

SOS repair requires activation of _

RecA protein

Explain SOS repair system response

1. RecA protein partially repressed by LexA protein

2. DNA damage activates RecA, in turn, activating LexA protease activity

3. LexA becomes degraded

   1. SOS response genes it represses become derepressed

      1. dinB: encodes error-prone DNA polymerase IV

      2. uvrA: encodes uvrA protein involved in error-free DNA repair

      3. umuCD: encodes umuCD protein involved in error-prone repair

      4. lexA: partially represses recA

SOS repair gene that partially represses RecA

LexA

SOS repair gene that encodes protein involved in error-free DNA repair

uvrA

SOS repair gene that encodes protein involved in error-prone DNA repair

umuCD

SOS repair gene that encodes error-prone DNA polymerase IV

dinB

• Lateral gene transfer

• Movement of genetic material between bacteria OTHER THAN by descent

Horizontal gene transfer

Sum of all the mobile genetic elements in a genome; not essential

Mobilome

T/F: Horizontal gene transfer involves genome replication but no cell division, unlike VGT which involves both.

TRUE

VGT = Genome replication, cell division
HGT = Genome replication

Transfer of genetic material by descent, i.e., from parent to offspring

Vertical gene transfer

Significance of HGT

1. Allows cells to quickly acquire new characteristics (e.g., metabolic diversity)

2. Provide source of nutrients (e.g., carbon, nitrogen, phosphorus)

Facilitates horizontal gene transfer

Mobilome

• Facilitates HGT

• Sum of all mobile genetic elements in a genome

Mobilome

5 types of mobile genetic elements

ppiti

• Plasmid

• Prophage

• Insertion sequence

• Transposon

• Integron

• Extrachromosomal circular dsDNA

• Genes (transferred) are not necessary for growth

Plasmid

Homologous vs. Nonhomologous recombination

• Homologous: requires similar sequences to integrate genetic material

• Nonhomologous: integrates genetic material into random locations in a genome

Identical sequences oriented in opposite directions

Inverted repeats

Catalyzes nonhomologous recombination

Transposase

• Integrated viral genomes

• e.g., Lysogenic bacteriophage

Prophage

Which MGE

Insertion sequence

Facilitates movement of transposable elements; recognizes and binds to inverted repeats

Transposase

Which MGE

Transposon

Can integrate viral DNA into host’s genome

Lysogenic bacteriophage

• Larger than insertion sequence but similar in function

• Contains multiple genes (conjugation and other functions)

Transposon

Transposons moves between different host DNA molecules through _

Transposase

2 types of transposition

• Conservative transposition

  • Transposon is excised from donor

  • Product

    • Donor DNA with break

    • Transposon in new location

  • Constant number of transposons bc donor loses transposition genes

• Replicative transposition

  • Transposon is replicated

  • Product

    • Undamaged donor DNA

    • Transposon in new location

  • Increase in transposons

• Simple mobile elements with tranposase gene (coding for transposase crucial to its mobility, cutting & inserting sequence from 1 location to another)

• Flanked by inverted repeats (recognized by transposase, which catalyzes nonhomologous recombination)

Insertion sequence

T/F: Conservative transposition maintains a constant number of transposable elements in the genome, as the original element is removed when the new copy is inserted.

TRUE

• Protein-coding genes

• DNA sequences with potential to be translated into protein

Open reading frame (ORF)

Often found in other mobile elements (e.g., plasmids, transposons)

Integron

Identical sequences oriented in same direction

Direct repeats

4 components of integron, explain image

1. Integrase (intl gene): catalyzes site-specific recombination of DNA at integron recombination site

2. Gene cassette: consists of 1 or more genes flanked by a series of recombination sites

3. Promoter (P_c): drives expression of genes within cassette

   1. *P_intl = promoter for integrase gene

4. Recombination site (attI, attC): contains direct repeats

*ORF1, ORF2 = open reading frames (DNA sequences with potential to be translated into protein; protein-coding genes)

T/F: Inserting a transposon into a gene can render it nonfunctional.

TRUE

Inserting transposon into a gene disrupts its coding sequence or regulatory elements, potentially rendering it nonfunctional. This gene inactivation method is actually used by researchers to study gene function and the effects of gene inactivation on the organism.

Mechanisms of HGT

• Transformation

• Transduction

• Conjugation

• Membrane vesicle-mediated HGT

Cells that are able to take up DNA and be transformed

Competent cell

How to make cells competent in the lab (or increase their efficiency)

Since DNA is negatively charged, cells can be made more positive through alteration of its surface properties using NaCl or sucrose, for instance, to make it more efficient at attracting DNA.

Naked DNA could either be _

• Cells free

• DNA fragment from medium

Uptake by competent cell of naked DNA (cells free or DNA fragment from medium)

Transformation

Refers to degree by which competent cell can readily and efficiently uptake DNA

Efficiency

High-efficiency bacteria

Bacillus

Poor-efficiency bacteria

Escherichia coli

Explain transformation in Gram (-) bacteria

1. Pilus extending from IM to extracellular environment to bind to extracellular DNA

2. Pilus retracting to bring in DNA into periplasm

*Type IV pili

Explain transformation in Gram (+) bacteria

1. Binding of DNA

   1. Recipient cell binds to naked DNA through help of DNA-binding proteins on its surface

2. Uptake of single-stranded DNA

   1. Nucleases break down one of strands of dsDNA

   2. Remaining strand is taken into recipient cell while being protected by single-stranded DNA binding proteins

3. RecA-mediated homologous recombination

   1. If incoming DNA is homologous, RecA protein facilitates its integration into recipient chromosome via homologous recombination

During transformation in gram(+), what protects the DNA from nuclease attack until it reaches the recipient’s chromosome?

Single-stranded DNA binding proteins (ssDBPs)

Transfer of genetic material via direct cell-to-cell contact (mating)

Conjugation

T/F: Conjugation is a plasmid-encoded mechanism

TRUE

What structure facilitates conjugation?

Conjugation pilus

T/F: Conjugation can mediate DNA transfer between closely related cells only.

FALSE

Conjugation can mediate DNA transfer between closely related cells or between more distantly related cells

Unidirectional transfer of genetic material

Conjugation

Give example of conjugation, explain

Fertility plasmid transferred via conjugation (unidirectional transfer for F+, F-)

• Donor strain = F+, Hfr

  • F+ = nonintegrated F plasmid

  • Hfr = F plasmid integrated into chromosome

    • High frequency of recombination

• Recipient = F-

T/F: If donor cell is F+, its recipient will become a donor. If donor cell is Hfr, its recipient remains a recipient.

TRUE

The entire F plasmid is sometimes too large to be fully transferred and is thus cut off in the case of Hfr recipient.

T/F: The recipient becomes Hfr only if the entire F plasmid, including the chromosomal region where it is integrated, is transferred

TRUE

Explain general process of conjugation

• Pilus of donor cell makes specific contact with receptor on recipient cell

• Pilus retracts by disassembling its units, pulling 2 cells together

• Donor and recipient remains in contact by binding coupling proteins in outer membrane of each cell

• DNA is transferred from donor to recipient through conjugation junction

Explain conjugation of PLASMID DNA

1. Pilus retracts

2. Cells are stabilized; F plasmid nicked in 1 strand

3. Transfer of 1 strand from F+ to F-

   1. Simultaneous replication of F plasmid in F+

4. Synthesis of complementary strand begins in recipient cell

5. Complete DNA transfer & synthesis. Cells separate

Explain transfer of CHROMOSOMAL DNA via conjugation

1. F plasmid nicked in 1 strand

2. F plasmid is transferred, followed by chromosomal DNA

3. Synthesis of second strand in both donor and recipient cells

Site where DNA transfer begins during conjugation

oriT (origin of transfer)

• Gene transfer mediated by phages (transducing particles) containing bacterial DNA (donor genes)

• Does not require cell-to-cell contact

• Unidirectional (from phage to transductant)

Transduction

• Host genes are occasionally and randomly packaged in phage head

• Lytic phage

• Low-frequency transfer: bc only a few phage particles will contain bacterial DNA

Generalized transduction

• Transfers very specific genes

• Occurs only with temperate and lysogenic viruses

• Extremely efficient and selective transfer

Specialized transduction

Phage DNA integrated into host genome

Prophage

When portion of host DNA is exchanged for phage DNA

Specialized transduction

T/F: In specialized transduction, only bacterial genes near the prophage integration site are transferred because the excision process is not random.

TRUE

Why generalized transduction is of low frequency compared to specialized?

Because generalized transduction occurs randomly and thus not every phage it produces will contain the viral DNA, while specialized transduction forms lysogenic phages, all of which with viral DNA integrated.