DNA repair Of the thousands of random changes created every day in the DNA of a human cell by heat, metabolic accidents, radiation of various sorts, and exposure to substances in the environment, only a few accumulate as mutations in the DNA sequence.

DNA repair is essential for all organisms. Because the survival of the individual demands genetic stability, analysis of the genomes of bacteria and yeasts has revealed that several percent of the coding capacity of these organisms is devoted solely to DNA repair functions.

What causes DNA damage? Thermal fluctuations Ultraviolet radiation Alkylating agents Gamma rays and X-rays

DNA damage delays progression of the cell cycle Cells will delay progression of the cell cycle until DNA repair is complete. For example, E. coli sulA protein is an inhibitor of cell division and it is expressed in response of SOS signal. Human ATM protein is a protein kinase that will send signals in response of oxygen-inflicted DNA damage.

Thermal fluctuations result in depurination of A and G Depurination (hydrolysis of the N-glycosyl linkages to deoxyribose) of A and G happen about 5,000 times every day per cell (in total).

Thermal fluctuations also cause deamination of cytosine Deamination of cytosine produces uracil, a base that will not appear in DNA. Deamination happens about 100 times every day per cell.

Deamination also happens to other bases Deamination of A (producing hypoxanthine) and G (producing xanthine) produces bases that will not occur in DNA naturally, therefore they will be easily recognized.

Ultraviolet radiations Ultraviolet radiations can produce a covalent linkage between two adjacent pyrimidine bases in DNA to form pyrimidine dimers.

Thymidine dimers Thymidine dimers are one of the products produced on DNA irradiated by ultraviolet radiation.

Alkylation Alkylating agents like EMS (ethylmethane sulfonate) are electrophiles. They seek center of negative charge (for example, DNA) and attack them by adding alkyl groups (carbon-containing groups).

Results of DNA damages left unrepaired Unrepaired DNA damages can lead to deletion of one or more bases pairs (in the case of depurination) or to a base-pair substitution in the daughter DNA chain (in the case of deamination and alkylation) when they are being replicated.

Unrepaired depurination leads to deletion

Unrepaired deamination leads to base-pair substitution

Why most of the organisms have double-stranded DNA as their genome? For safe storage of genetic information, double-stranded DNA is the best choice for genome. When one strand is damaged, the complementary strand retains an intact copy of the same information, and the copy is generally used to restore the correct nucleotide sequences to the damaged strand.

Two common pathways for DNA repair Base excision repair - DNA glycosylases + AP endonucleases - AP endonucleases Nucleotide excision repair

Base excision repair Base excision repair involves a battery of DNA glycosylases. There are at least six types of DNA glycosylases: - removing deaminated Cs and As - removing different types of alkylated or oxidized bases - removing bases with opened rings - removing bases in which a C=C has be converted to C-C - removing T from T-G pair (inefficiently)

Mechanisms of DNA glycosylases action DNA glycosylases flip out base while travel along DNA to find out the altered nucleotide from the helix.

Mechanisms of DNA glycosylases action Once a damaged base is recognized, the DNA glycosylase creates a deoxyribose sugar that lacks the base (AP site).

After DNA glycosylases create AP site, AP endonucleases join AP endonucleases recognize the AP site, cut the phosphodiester backbone, leave a gap.

After AP endonucleases The gap is then repaired by DNA polymerase and DNA ligase.

AP endonucleases are also involved in repairing depurinated DNA Depurination also leaves an AP site. Depurinated DNA will be directly repaired by AP endonuclease as described previously.

Nucleotide excision repair Nucleotide excision repair can repair almost any large change in the structure of the DNA double helix. A large multienzyme complex scans the DNA for a distortion in the double helix, rather than for a specific base change.

Nucleotide excision repair Once a bulky lesion has been found, the phosphodiester backbone of the abnormal strand is cleaved on both sides of the distortion.

Nucleotide excision repair The oligonucleotide containing the lesion is peeled away from the DNA double helix by DNA helicase. DNA polymerase and ligase will repair the large gap.

Deamination problems Although most of the deamination products are unnatural bases and will be recognized by repair machinery…

Deamination problems Deamination of methylated C (which happens a lot in inactivated genes) produces thymine, a naturally occurring DNA. Although a special DNA glycosylase recognizes a mismatched base pair involving T in the sequence T-G and removes the T, but this enzyme is rather ineffective. As a result, 1/3 of the single base mutations comes from these methylated nucleotides!

Gamma rays and X-rays While all the other factors damage DNA by modifying nucleotides, gamma rays and X-rays damage DNA by breaking one or both strands. Single-strand breaks are easily repaired. However, double-strand breaks will result in chromosome degradation if left unrepaired.

Repairing double-strand breaks Double-strand breaks can be repaired either by nonhomologous end-joining (NHEJ) or homologous end-joining.

Nonhomologous end-joining Broken ends are juxtaposed (placed side- by-side) and rejoined by DNA ligation, generally with the loss of one or more nucleotides at the site of joining.

Homologous end-joining Homologous end-joining requires special recombination proteins that recognizes areas of DNA sequence matching between the two chromosomes and bring them together.

Homologous end-joining A DNA replication process then uses the undamaged chromosome as the template for transferring genetic information to the broken chromosome, repairing it with no change in the DNA sequence.

Homologous end-joining Homologous end-joining is the major route for repairing DNA double-strand breaks in bacteria, yeasts, Drosophila, and also in those organisms which little nonhomologous end-joining is observed.

SOS response – the error-prone bypass In E. coli, an excess of single-strand DNA induce the expression of more than 15 genes. They will not only increase cell survival after damage but also increase the mutation rate by increasing the number of errors made in copying DNA sequences. This is due to the usage of low-fidelity DNA polymerases.

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UmuD’ UmuD’2C (DNA pol V)

Why use low-fidelity DNA polymerase? Because only low-fidelity DNA polymerase can efficiently use damaged as template for DNA synthesis, so it is employed during SOS response. Human cells contain more than ten minor DNA polymerases, many of which are error-prone.

General recombination General recombination is one type of the genetic recombination.

General recombination General recombination is also called homologous recombination. Homologous end-joining is involved in general recombination. General recombination is essential for every proliferating cells, because accidents occur during nearly every round of DNA replication that interrupt the replication fork and require general recombination mechanisms to repair. Cross-over during meiosis also use the same mechanism of general recombination.

General recombination General recombination creates DNA molecules of novel sequence because the heteroduplex joint can tolerate a small number of mismatched base pairs. Usually these two DNA molecules are not exactly the same on either side of the joint.

General recombination mechanism (1) DNA helix is broken by special endonuclease (for example, HO endonuclease in S. cerevisiae).

General recombination mechanism (2) Limited degradation of the 5’ end is executed by exonuclease. RecA from E. coli, a SSB and DNA-dependent ATPase, is essential in this part to keep ssDNA in extended conformation.

RecA is not only essential in DNA repair but also in recombination. RecA has more than one DNA-binding site so it can hold a single strand and a double helix together. RecA

RecA in general recombination With RecA, the region of homology is identified before the duplex DNA target has been opened up, through a three-stranded intermediate in which the DNA single strand forms transient base pairs with bases that flip out from the helix in the major groove of the double-stranded DNA molecule.

General recombination mechanism (3): strand invasion and branch migration The unpaired region of one of the single strands displaces a paired region of the other single strand, moving the branch point without changing the total number of DNA base pairs.

RecA protein guides the direction of branch migration -ATP The ATP-bound form of RecA binds to DNA tighter than ADP-bound form. New RecA molecule (RecA-ATP) are preferentially added at one end of the RecA protein filament then ATP hydrolysis followed. -ATP -ADP -ATP -ATP -ATP -ATP -ADP -ADP -ATP -ATP -ATP -ATP -ATP

RecA protein is essential The homolog of RecA protein in human (Rad51) is also essential. These proteins require accessory proteins to help them functioning properly. Two of the hRad51 accessory proteins, Brca1 and Brca2, are related to breast cancer.

General recombination mechanism (4) At this stage, DNA replicating machines use the 3’ end as primer to start DNA synthesis with the pairing strand (the other DNA duplex) as template. Gene conversion also happens during this stage.

Gene conversion

Results of gene conversion

General recombination mechanism (5) Holliday junction is formed after the other end is paired and DNA synthesis is completed.

Holliday junction Holliday junction (cross-strand exchange) is the key recombination intermediate. After it is formed, it can isomerize to the open structure (B) or further isomerization will make the crossed strand becoming non-crossing strand.

The open structure of Holliday junction When the open structure of Holliday junction is formed, two RuvB hexamer and one RuvA tetramer will engage the resolution of Holliday junction, producing recombined DNA molecules.

RuvA tetramer Two RuvB hexamer use energy from ATP hydrolysis to extend the heteroduplex region by moving out the open circle.

General recombination mechanism (6) With help of RuvA, RuvB and other proteins, selective strands were cut and ligated and exchanged DNA with heteroduplex region of several thousand base paired were formed.

General recombination leads to different products in mitotic and meiotic cells Guided by specific proteins 99% mitotic cells go this way

Proteins involved in mismatch repair is also involved in general recombination mutL mutS (mutH)