MB 207 – Molecular Cell Biology DNA Damage and Repair DNA recombination

DNA Damage and Repair Maintaining genetic stability is very important - accurate mechanism for replicating DNA. - mechanism for repairing DNA alterations that arise both spontaneously and from exposure to DNA-damaging environmental agents. Nearly all DNA damage is harmful but occasionally beneficial because mutations provide genetic variability. How important is DNA repair? DNA is the only biomolecule that is specifically repaired. All others are replaced. DNA damage is repaired shortly after it occurs and hence it does not affect future generations >100 genes participate in various aspects of DNA repair, even in organisms with very small genomes. Cancer is caused by mutations as well as many other diseases.

Spontaneous mutations Hydrolysis reactions caused by random interactions between DNA and the molecules around it. Two types of spontaneous mutations: Depurination Deamination Depurination the loss of a purine base by spontaneous hydrolysis of glycosidic bond that links it to deoxyribose. this glycosidic bond is labile under physiological conditions. Therefore, susceptible to hydrolysis that DNA loss thousands of purine bases in the human cell everyday.

Deamination Primary amino groups of nucleic acid bases are unstable. They can be converted to keto groups in the hydrolysis reactions and become deaminated. Involve cytosine, adenine and guanine, changes the base pairing properties of the affected base. Cytosine is more susceptible to deamination, giving rise to uracil. Others: Adenine to Hypoxanthine, Guanine to Xanthine, and 5-methyl cytosine to Thymine. Usually caused by random collision of a water molecule with the bond that links the amino group of the base to the pyrimidine or purine ring. Rate is about 100 deaminations per day. If not repaired, the error base sequence may be propagated when the strand serves as a template in the next round of replication.

Depurination & Deamination

Deamination of DNA nucleotides

A. Deamination of cytosine B. Depurination A. Deamination of cytosine produces uracil Missing purine Results in the substitution of one base for another when the DNA is replicated If uncorrected, can lead to either the substitution or the loss of a nucleotide pair.

Mutagens (mutation-causing agents) Two major categories Chemicals Radiations - alter DNA structure by a variety of mechanisms. Base analogs - resemble nitrogenous bases in structure and are incorporated into DNA. Base modifying agents - reacts chemically eith DNA bases to alter their structures. Intercalating agents - insert themselves between adjacent bases of the double helix.

Radiations Sunlight (ultraviolet radiation) - alters DNA by triggering pyrimidine dimer formation (formation of covalent bonds between adjacent pyrimidine bases). - blocked replication and transcription. X-rays and related form of radiation emitted by radioactive substances - ionizing radiation because it removes electrons from biological molecules. - generating highly reactive intermediates that cause various types of DNA damage.

DNA damages Distortion of double helix structure Strand breaks The thymine dimer Distortion of double helix structure Photodamage UV light absorbed by the nucleic acid bases can induce bond formation between adjacent pyrimidines (C or T) within one strand. The two adjacent pyrimidines are pulled closer to each other than in normal DNA Strand breaks Single-strand and double-strand breaks are produced at low frequency during normal DNA metabolism by topoisomerases, nucleases and repair processes as well as by ionizing radiation. This type of damage is introduced into DNA in cells that are exposed to ultraviolet irradiation

Types of DNA damages hydrolytic attack (blue arrows) Spontaneous oxidative damage (red arrows) hydrolytic attack (blue arrows) Uncontrolled methylation (green arrows))

Repairing Damaged Bases Damaged or inappropriate bases can be repaired by several mechanisms: Direct chemical reversal of the damage Excision Repair, in which the damaged base or bases are removed and then replaced with the correct ones in a localized burst of DNA synthesis. There are three modes of excision repair, each of which employs specialized sets of enzymes. Base Excision Repair (BER) Nucleotide Excision Repair (NER) Mismatch Repair (MMR) 3. Double strand breaks Non-homologous end joining Homologous recombination 4. Translesion synthesis- DNA damage tolerance process that allows the DNA replication machinery to replicate past DNA lesions such as thymine dimers or AP sites

Direct Reversal of Base Damage The most frequent cause of point mutations in humans is the spontaneous addition of a methyl group (CH3-) (an example of alkylation) to Cs followed by deamination to a T. Fortunately, most of these changes are repaired by enzymes, called glycosylases, that remove the mismatched T restoring the correct C. This is done without the need to break the DNA backbone (in contrast to the mechanisms of excision repair described below). Some of the drugs used in cancer chemotherapy ("chemo") also damage DNA by alkylation. Some of the methyl groups can be removed by a protein encoded by our MGMT gene. However, the protein can only do it once, so the removal of each methyl group requires another molecule of protein. This illustrates a problem with direct reversal mechanisms of DNA repair: they are quite wasteful.

2. Excision Repair Base excision repair (BER), which repairs damage to a single nucleotide caused by oxidation, alkylation, hydrolysis, or deamination. The base is removed with glycosylase and ultimately replaced by repair synthesis with DNA ligase. B. Nucleotide excision repair (NER), which repairs damage affecting longer strands of 2–30 bases. This process recognizes bulky, helix-distorting changes such as thymine dimers as well as single-strand breaks (repaired with enzymes such UvrABC endonuclease). A specialized form of NER known as Transcription-Coupled Repair (TCR) deploys high-priority NER repair enzymes to genes that are being actively transcribed. C. Mismatch repair (MMR), which corrects errors of DNA replication and recombination that result in mispaired (but normal, that is non- damaged) nucleotides following DNA replication

DNA repair mechanisms 1) Base excision repair (BER) Removal of the incorrect base by an appropriate DNA glycosylase to create a deoxyribose sugar lacking it’s base (AP site - apurinic / apyrimidinic) Nicking of the damaged DNA strand by AP endonuclease upstream of the AP site, thus creating a 3'-OH terminus adjacent to the AP site, removal of sugar phosphate. Extension of the 3'-OH terminus by a DNA polymerase, DNA ligase seals nick. e.g. removal of uracil from DNA

DNA repair mechanisms 2) Nucleotide excision repair (NER) (bulky lesion) 2) Nucleotide excision repair (NER) Removes a whole oligonucleotide that contain the damage. Steps: Multienzyme complex recognizes damaged regions based on their abnormal structure as well as on their abnormal chemistry (eg. pyrimidine dimer) Double incision of the damaged strand several nucleotides away from the damaged site, on both the 5' and 3' sides An associated DNA helicase removes the entire damaged strand, in-between the nicks. Bacteria multienzyme complex leaves a 12nt gap; doubles the size in human DNA Filling in of the resulting gap by a DNA polymerase Ligation by DNA ligase.

Repairing Strand Breaks Ionizing radiation and certain chemicals can produce double-strand breaks (DSBs) in the DNA backbone. Double-Strand Breaks (DSBs) There are two mechanisms by which the cell attempts to repair a complete break in a DNA molecule: i. Direct joining of the broken ends. -This requires proteins that recognize and bind to the exposed ends and bring them together for ligasing. They would prefer to see some complementary nucleotides but can proceed without them so this type of joining is also called Nonhomologous End-Joining (NHEJ). -Errors in direct joining may be a cause of the various translocations that are associated with cancers. (Translocation: Type of mutation in which a portion of 1 chromosome is broken off and attached to another)

ii. Homologous Recombination ii. Homologous Recombination. Here the broken ends are repaired using the information on the intact -sister chromatid (available in G2 after chromosome duplication), or on the -homologous chromosome (in G1; that is, before each chromosome has been duplicated). This requires searching around in the nucleus for the homolog — a task sufficiently uncertain that G1 cells usually prefer to mend their DSBs by NHEJ. or on the -same chromosome if there are duplicate copies of the gene on the chromosome oriented in opposite directions (head-to-head or back-to-back). -Two of the proteins used in homologous recombination are encoded by the genes BRCA1 and BRCA2. Inherited mutations in these genes predispose women to breast and ovarian cancers.

DNA repair mechanisms Two different types of end-joining for repairing double-strand breaks 1. Nonhomologous end-joining permits joining of double-strand breaks even if there is no sequence similarity between them Broken ends are rejoined by DNA ligation with the loss of one or more nucleotides at the joining site Alters the original DNA sequence either by deletions or short insertions. 2. Homologous end-joining More difficult to accomplish but is more precise cells are diploid – contain 2 copies of each double helix Recombination mechanisms used to transfer nucleotide sequence information from the homologous intact DNA double helix to the site of the double-strand break Both system involve a lot of different proteins and the processes are much more complicated

DNA end-joining for repairing ds breaks Accidental break (ionizing radiation, oxidizing agents, replication errors) Loss of nucleotides due to degradation from ends DNA ligation Copying process involving homologous recombination Complete sequence restored by copying from second chromosome (replication process uses the undamaged chromosome as the template for transferring genetic information to the broken chromosome, repairing it with no change in the DNA sequences) Region with altered segment due to missing nucleotides Nonhomologous end-joining - Common in mammalian cells Homologous end-joining

Summary of DNA repair systems Type Damage Enzyme Mismatch repair Replication errors MutS, MutL, and MutH in E. coli MSH, MLH and PMS in humans Photoreaction Pyrimidine dimers DNA photolyase Base excision repair Damaged base DNA glycosylase Nucleotide excision repair Pyrimidine dimer Bulky adduct on base UvrA, UvrB, UvrC and UvrD in E. coli XPC, XPA, XPD, ERCI-XPF and XPG in humans Double strand break repair Double strand breaks RecA and RecBCD in E.coli Translesion DNA synthesis Pyrimidine dimer or apurinic site Y-family DNA polymerase, such as UmuC in E. coli

DNA Recombination A process that a DNA segment moves from one DNA molecule to another DNA molecule DNA molecules recombine by breaking and rejoining Phosphodiester bonds are broken and rejoined. Importance of DNA recombination: the process of introducing genetic variation: Genetic variation is crucial to allow organisms to evolve in response to a changing environment. E.g., genetic recombination results in the exchange of genes between paired homologous chromosomes during meiosis. an important mechanism for repairing damaged DNA. involved in rearrangements of specific DNA sequences that alter the expression and function of some genes during development and differentiation. Two broad classes are commonly recognized - general recombination & site-specific recombination.

General recombination in meiosis A heteroduplex joint General recombination in meiosis

General Recombination Allow large section of the DNA double helix to move from one chromosome to another Responsible for the crossing-over of chromosomes during meiosis Chromosome must synapse (pair) in order for chiasmata to form where crossing-over occurs DNA synapsis: base pairing between complementary strands from 2 DNA molecules Chiasmata: regions where paired homologous chromosomes exchange genetic material during meiosis, a cross-shaped structure Only occurs between homologous DNA molecules

General Recombination Two homologous DNA molecules line up. Nicks (single or double??) are introduced. Each nicked strand then invades the other DNA molecule by complementary base pairing. The cut strands cross and join homologous strands, forming the Holliday structure (or Holliday junction) (R. Holliday (1964). Once a Holliday junction is formed, it can be resolved 2 ways by nicking and rejoining of the crossed strands to yield 2 different heteroduplexes: recombinant  heteroduplexes: resulting DNA molecules are a combination of both parental DNA molecules. non-recombinant heteroduplexes: resulting DNA molecules contain only DNA from one parent molecule with a small portion of heteroduplex.

Holliday junction cleavage Paternal chromosome A Maternal chromosome B DNA clearage ‘splice’ or crossover products reassortment or flanking genes ‘patch’ or non- crossover products no reassortment DSB repair model for homologous recombination. The figure shows the step leading to generation of recombination intermediate with 2 Holliday junctions. Recombinant Non-recombinant

General Recombination: example Various enzymes (homologues) are involved in the recombination process: Rec A: catalyze the exchange of strands between homologous DNAs that causes heteroduplexes to form RecB, C, & D: complex of three proteins acts as a helicase and transiently unwinds the double-stranded DNA When it encounters the specific nucleotide sequence GCTGGTGG (the chi site), the enzyme acts as a nuclease to introduce a single-stranded nick Continue to unwind the double helix, forming a displaced single strand to which RecA can bind to initiate strand exchange. Ruv A, B: catalyze the movement of the crossed-strand site in Holliday junctions RuvC: resolves the Holliday junction by cleaving the crossed strands, which are then joined by ligase

The different resolutions of a general recombination intermediate in mitotic and meiotic cells.

Site-Specific Recombination Occurs between sequences with a limited stretch of similarity; involves specific sites Mediated by proteins that recognize the specific DNA target sequences rather than by complementary base pairing Transposons/transposable elements/ “Jumping genes“: mobile genetic elements that can move throughout the genome Two distinct mechanisms: 1. Transpositional site-specific recombination: insertion of mobile genetic elements into any DNA sequence, no formation of heteroduplex 2. Conservative site-specific recombination: site specific recombination that requires a short DNA sequence that is the same on both donor and recipient, involve formation of heteroduplex

Transposon (cont’ next) Transposase / Integrase: act on the specific sequence at the end of transposon and disconnecting it from the flanking sequence and then inserting it to a new target site

Cut and Paste transposition (DNA-only transposons ) Steps of cut and paste transposition: Binding of transposase subunits to the terminal inverted repeats Transpososome formation (synaptic complex) Excision of the transposon (contrast to replicative mechanism) DNA strand transfer Gap repair – DNA polymerase

Replicative transposition (DNA-only transposons) Steps of Replicative transposition mechanism: Binding of transposase protein to transposon sequence. Transposon DNA is replicated and a copy is inserted at a new chromosomal site, leaving the original chromosome intact. nick

The life cycle of a retrovirus

Retrovirus-like transposition LTR on the two ends of the element Transcription to generate RNA copiy RNA template to synthesize DNA using reverse transcriptase cDNA is recognized by integrase Gap repair

Non-retroviral transposition -poly-A retrotransposons move by a ‘Reverse Splicing’ mechanism called target site primed reverse transcription A significant fraction of vertebrate chromosomes is made up of repeated DNA sequences In human chromosomes, these repeats are mostly mutated/truncated versions of a retrotransposon called L1 element (LINE= lone interspersed nuclear element) L1 element are mostly immobile Translocation result in human disease eg. Hemophilia – L1 insertion into a gene for blood clotting factor VIII. Mechanism: require a complex of endonuclease and a reverse transcriptase

Non-retroviral transposition Generates a ssDNA element directly linked to target DNA Processing of ssDNA to produce dsDNA of L1

Conservative site-specific recombination Breaking and joining occur at two special sites, one on each participating DNA molecules enzymes involve can break and rejoin two DNA helix, often reversible, ie. DNA integration, DNA excision or DNA inversion can occur eg. Bacteriophage lambda/ bacterial viruses – mobile DNA element, moving in and out of host chromosomes eg. Bacteriophage eg. Salmonella typhimurium Inversion of DNA segment changes the type of flagellum produced

Conservative site-specific recombination Insertion of a circular bacteriophage lambda DNA chromosome into bacteria chromosome: Integrase binds to specific ‘attachment site’ on each chromosome 2. Cuts and switch the partner strands 3. Re-seals forming heteroduplex joint (7nt bp long) 4. Phosphodiester bond breakage release energy used for strand joining 5. Intergrase dissociates.

a specific gene in a group of cells in a transgenic animal: How a conservative site-specific recombination enzyme is used to turn on a specific gene in a group of cells in a transgenic animal: (used in mice or Drosophila to study the effect of expressing a gene of interest in the animal, using Cre recombination enzyme & loxP recognition sites)

Major types of transposable elements Structural features Mechanism of movement DNA-mediated transposition Bacterial replicative transposons Terminal inverted repeats that flank antibiotic resistance and transposase genes Copying of element DNA accompanying each round of insertion into a new target site Bacterial cut and paste transposons Excision of DNA from old target site and insertion into new site Eukaryotic transposons Inverted repeats that flank coding region with introns RNA-mediated transposition Viral-like retrotransposons ~250 to 600bp direct terminal repeats (LTRs) flanking genes for reverse, transcriptase, integrase and retroviral-like Gag protein Transcription into RNA from promoter in left LTR by RNA polymerase II followed by reverse transcription and insertion at target site Poly-A retrotransposons 3’ A-T rich sequence and 5’ UTR flank genes encoding an RNA-binding protein and reverse transcriptase Transcription into RNA from internal promoter; target primed reverse transcription initiated by endonuclease cleavage