DNA Repair and Recombination
Model of double strand break repair for homologous recombination during meiosis.
1) a double-strand break in one of two homologs is converted to a double-strand gap by the action of exonucleases. Strands with 3' ends are degraded less than those with 5' ends, producing 3' single-strand-extensions. 2) An exposed 3' end pairs with its complement in the intact homologs. The other strand of the duplex is displaced. 3) The invading 3' end is extended by DNA polymerase plus branch migration, eventually generating a DNA molecule with two crossover called Holliday intermediates. 4) Further DNA replication replaces the DNA missing from the site of the original double-strand break. 5) Cleavage of the Holliday intermediates by specialized nucleases generates either noncrossover or crossover recombination products.
How can new DNA be introduced into a bacterial cell all
1) conjugation during bacterial mating 2) transformation, when DNA is taken up by cells 3) transduction, when bacterial DNA that was packaged into a phage particle is introduced by infection.
What is the function of homologous/ general recombination
1) contributes to the repair of several types of DNA damage. 2) It provides, in eukaryote cells, a transient physical link between chromatids that promotes an orderly segregation of chromosomes at the first meiotic cell division. 3) it enhances genetic diversity in a population.
What are the most prominent endogenous DNA-damaging reactions (common sources of spontaneous mutation)
1) depurination 2) deamination 3) oxidation 4) nonenzymatic methylation by S-adenosylmethionine.
What is the Holliday Model of General Recombination
1) recombination begins with nicking at the same site on two paired chromosomes. 2) partial unwinding of the duplexes is followed by strand invasion, in which a free single-strand end from one duplex pairs with its unbroken complementary strand in the other duplex, and vice versa. 3) Holliday junction: enzymatic ligation generates a crossed-strand intermediate. 4) branch migration: the crossed- strand structure can move in either direction by duplex unwinding and rewinding. 5) the process leading to recombination begins with isomerization (lower strand rotate 180º/ left ends rotate 180º) of the Holliday structure. 6) single breakage and sealing at crossover point.
What is the optimal wavelength for DNA photoylase to work in
370nm most effective.
Excision repair involves
endonuclease cleavage on both sides of a damaged site, followed by replacement synthesis. Polymerase I Base excision repair. Nucleotide excision repair.
What is DNA photolyase (photoreactivating enzyme)
enzyme that repairs cyclobutane pyrimidine dimers in the presence of visible light.
What are Thymine dimers
formed from two adjacent DNA thymine residues.
What is homologous recombination
genetic exchanges between any two DNA molecules (or segments of the same molecule) that share an extended region of nearly identical sequence. Occurs between pieces of DNA that have closely related sequences.
What is silent mutation
if the mutation affects nonessential DNA or if it has a negligible effect on the function of the gene.
What are the environmental DNA-damaging agents
ionizing radiation ultraviolet radiation DNA-methylating reagents such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). DNA-cross-linking reagnets such as the anticancer drug cisplatin. Bulky electrophilic agents, such as benzo[a]pyrene, one of the carcinogenic hydrocarbons in tobacco smoke.
What does isomerization do to the strand
lower strand rotates 180º. left ends rotate 180º.
What is daughter- strand gap repair
newly replicated DNA duplexes undergo genetic recombination, with ultimate removal of the damaged DNA segment
What is nonhomologous recombination
occurs between unrelated sequences (e.g. Transponsons)
What is oxidation
oxidation of guanine to 8-oxoguanine or of thymine to thymine glycol
How do cells protect DNA damage due to natural errors
polymerase base selection. proofreading. mismatch repair
What are the proteins that participate in mismatch repair in E.coli
products of genes mutH, mutL, and mutS. also require the gene product MutU (DNA helicase II)
Recombination & evolution
recombination enzymes probably evolved to help DNA repair (confers a selective advantage). Recombination also creates new combinations of genes on a chromosome, increasing changes of evolutionary survival.
What is Base Excision repair (BER)
remove one or more nucleotide from a site of base damage. This process initiates with enzymatic cleavage of the glycosidic bond between the damaged base and deoxyribose.
What causes mismatches
replication errors, through deamination of 5-methylcyotsine in DNA to yield thymine, or through recombination between DNA segments that are not completely homologous. mismatches result when DNA polyperase slides along its template, creating short loops or bulges in duplex DNA.
What is double-strand break repair
several ends of a duplex can be rejoined.
What is deamination
usually the hydrolytic conversion of a DNA-cytosine residue to uracil. DNA glycosylases hydrolyze base-sugar N-glycosidic bond. Deaminated bases are removed and replaced.
What is Holliday junction
An intermediate formed during recombination between two double stranded DNA molecule. Enzymatic ligation generates a crossed-strand intermediate.
What is the biological significance of thymine dimers
Biologically significant photoproducts because the relative abundance of thymine dimers in irradiated DNA correlated most closely with death of irradiated phages or bacteria. Thus, the ability of an organism to survive UV irradiation was related directly to its ability to remove thymine dimers from its DNA.
What is translesion synthesis (error-prone repair)
DNA damage that blocks replicative DNA polymerases can be copied by specialized byt often inaccurate, DNA polymerase
Repair of Damaged DNA
DNA is the only cellular macromolecule that can be repaired.
What is the general excision-repair pathway
Excision-repair enzymes detect damaged DNA. An endonuclease nicks the DNA backbone on both sides of the damage. A helicase or exonuclease removes the damaged DNA, leaving a gap. DNA polymerase fills the gap. The remaining nick is sealed by DNA ligase
What are photoproducts
In UV-irradiated DNA or DNA extracted from UV-irradiated organisms, small amounts of many different altered DNA constituents. Prominent among them are intrastrand dimers consisting of two pyrimidine bases joined by a cyclobutane ring structure involving C5 and 6
When do DNA recombination occur in meiosis
In prophase 1. Crossing over at chiasma
Why is DNA repair possible
The DNA molecule consists of two complementary strands. DNA damage in one strand can be removed and accurately replaced by using the undamaged complementary strand as template. DNA can be repaired directly, by changing a damaged base to a normal one, or indirectly, by replacing a DNA segment containing the damaged nucleotide.
How does DNA photolyase work
The enzyme binds to DNA in a light-independent process, specifically at the site of pyrimidine dimers. In the presence of visible-wavelength light, the enzyme catalzes chemical cleavage of the dimer, the bonds linking the pyrimidine rings are broken, thereby restoring normal base pairing and repairing the DNA. After which the enzyme can dissociate in the dark.
What happens in mismatch repair
The mismatch correction system scans newly replicated DNA, looking for both mismatched bases and single-base insertions or deletions. MutS binds to DNA at the site of the mismatch, followed by the binding of MutL and then MutH. The MutHLS complex moves along DNA in both directions until it encounters the nearest 5'-GATC sequence. An endonuclease activity of MutH then cleaves on the 5' side of the G in the unmethylated strand (use energy from ATP -> ADP + Pi). At that point helicase II unwinds the DNA, moving back past the mismatch, followed by an exonuclease that digests the displaced single strand - exonuclease VII or RecJ nuclease if the cut is 5' to the mismatch, and exonuclease I or X if the cut is on the 3' side. The resultant gap is filled by DNA polymerase III holoenzyme and DNA ligase, working in concert with SSB (single strand break). Methyl transferase then transfers methyl group to A on repaired daughter strand.
What is the function of MutS
MutS is a 'motor protein' which uses the energy of ATP hydrolysis to pull DNA from both directions until it reaches the site at which the repair process is to being. When it finds an appropriate signal, part of one strand containing the mismatched region is cut and replaced.
What is deamination of C
Mutation of cytosine (C) to uracil (U), changing the complementary base from Guanine (G) to Adenine (A), creating one normal copy and one mutated copy after DNA replication.
What are the sources of mutation
Natural polymerase error. Endogenous DNA damage: oxidative damage, depurination. Exogenous DNA damage: radiation, chemical adducts. "Error-prone" DNA repair
Strand exchange catalyzed by RecA
Rec A-coated strand binds to homologous double-stranded DNA. Strand invation and displacement occur. Branch migration extends the region of exchange.
Recombination in E.coli
Recombination starts with generation of single stranded DNA with a free 3' end. In E.coli, RecBCD endonuclease binds to DNA, cleaves one strand, then unwinds DNA in an ATP-dependent reaction. Strand exchange begins when single-stranded DNA (coated with RecA at the outset) invades a neighboring double helix. RecA is a strand exchange protein. Synapsis occurs 5' to 3' direction. RecA molecule is displaced after its associated DNA has base-paired with its complement. The displaced strand may wrap around the newly formed duplex.
What is formed if the strands that break are those that were not broken in 1).
Resolution of the resulting structure generates two chromosomes recombinant for DNA flanking the region and each containing a heteroduplex region (both strands are recombinants).
How can spontaneous hydrolysis result in deamination
Spontaneous hydrolysis removed NH2 from cytosine, replacing the NH2 with OH, forming uracil.
What is strand invasion.
a free single-strand end from one duplex pairs with its unbroken complementary strand in the other duplex, and vice versa.
What is RecBCD
a multifunctional enzyme, unwinds and rewinds DNA, with one strand being unwound more rapidly and then converted to a single-stranded 3' end.
What is mutation
a permanent change in the nucleotide sequence of DNA. Can involve the replacement of one base pair with another (substitution mutation) or addition or deletion of one or more base pairs (insertion or deletion mutations).
What is mismatch repair
a process that recognizes DNA mismatches created either by replication errors, nonhomologous recombination, or damage to one DNA base and corrects the error. To catch single-base errors that slip through proofreading during replication. Happens right after replication. Improves overall fidelity replication by a factor of 10^2 to 10^3
What is nucleotide excision repair
a section of DNA that contains a damaged site is excised and replaced with normal DNA. repairs damage due to environment mutagens such as UV rays
What is an apyrimidinic site
a sugar residue lacking an attached pyrimidine
DNA can be damaged by
alkylation, methylation, deamination, loss of heterocyclic bases (depurination or depyrimidization)
What is recombination
any process that involves the formation of new DNA from distinct DNA molecules (exchange or transfer), such that genetic information from each parental DNA molecule is present in the new molecule. end-to-end joining from two different DNA molecules.
How does cell protects from endogenous/ exogenous DNA damage
base excision repair, nucleotide excision repair, (recombination, polymerase bypass) recombination and polymerase pymass do not remove damage but remove its block to replication. Polymerase bypass itself is often mutagenic.
What does DNA damage include
base modification. nucleotide deletions or insertions cross-linking of DNA strands. breakage of phosphodiester backbone.
Double-helical DNA is very sensitive to
damage by UV light
What is direct repair
damaged DNA base undergoes a chemical or photochemical reaction to restore the original structure.DNA photolyases. photoactivation of cyclobutane pyrimidine dimers induced by UV. methyltransferase, repairs alkylated bases.
What is depurination of adenine (A)
depurination of adenine (A) (adenine removed, no pairing with thymine). during DNA replication, the A-T nucleotide pair has been deleted in one daughter DNA, whilst a normal daughter DNA is produced.
Why replication cannot proceed in the presence of pyrimidine dimers?
Dimerization draws the adjacent thymine residues together, distorting the helix such that replicative polymerization past this site is blocked.
What is common in a DNA strand
Dimerization of adjacent pyramidines (e.g. thymines)
What are the major repair mechanisms
Direct repair Excision repair Mismatch repair Double strand breaks repaired mainly by end-joining.
DNA repair enzymes include photoylase, which
uses light energy to repair pyrimidine dimers, and alkyltransferases. 'enzymes' that are inactivated after just one catalytic cycle.
How does DNA maintain its metabolic stability
The stability is maintained in two ways, by a replication process of high accuracy, and by mechanisms for correcting genetic information when DNA suffers damage.
What happens in photodimerization of adjacent thymines?
UV light cause covalent bond to form between adjacent thymidine residue along a DNA strand (cross-linked), distorting the helix. intrastrand dimers consisting of two pyrimidine bases (thymine) joined by a cyclobutane ring structure involving C5 and 6
What are the DNA repair mechanisms
specific repair enzymes scan DNA to detect any alterations. Repair protects individual cells and subsequent generations. But error could be replicated and carried to daughter cells.
How does spontaneous hydrolysis result in depurination
spontaneous hydrolysis removed the guanine from the nucleotide and is replaced with OH.
What is base excision repair
starts with cleavage fo teh glycosidic bond connecting a damaged base to the DNA supar-phosphate backbone. Remove damaged base.
Excision-repair pathway in E.coli
the 3-subunit UvrABC enzyme recognizes a lesion (a thymine dimer) and, with the help of ATP hydrolysis, forces DNA to bend, leading to cleavage of the damaged strand at two sites, leaving a potential gap eleven nucleotides in length, with a 3' hydroxyl group and a 5' phosphate at the end. Pilymerase and ligase action then replace the damaged 11-mer with undamaged DNA. Helicase II, the product of the uvrD gene, is also required to unwind and remove the excised oligonucleotide, which is ultimately broken down by other enzymes.
What is branch migration
the crossed- strand structure can move in either direction by duplex unwinding and rewinding.
What is depurination
the hydrolytic rupture of the glycosidic bond between deoxyribose and a purine base.
How does the mismatch repair system recognise the right strand to repair
the mismatch repair enzymes identify newly replicated strand because for a short period, that DNA is unmethylated. The mismatch repair enzymes look for -GATC- sequences that are not methylated. Recognition of an unmethylated GATC can target that strand for mismatch correction at a site as far as 1kp between the mismatch and the GATC site, in either direction. Once the methylation system has acted on all GATC sites in the daughter strand, it is too late for the mismatch repair system to recognise the more recently syntheiszed DNA strand, and any advantage in total DNA replication fidelity is lost.
What is transposition
the movement of DNA from one chromosomal integration site to another.
What is formed if the strands that break are those that were broken in 1)
the products are nonrecombinant duplexes, each containing a heteroduplex region. Only these strands are recombined.
Where is the site of methylation in E.coli after replication
the sequence GATC is crucial because this is the site methylated soon after replication, by action of teh product of the dam gene (DNA adenine methylase) - Dam methylase on the 6 position of the A residue in the GATC sequence
What are repaired in all organisms
thymine dimers
Replacement of Uracil in DNA by BER
uracil-DNA N-glcosylase hydrolytically cleaves the glycosidic bond between N-1 of uracil and C-1 of deoxyribose (N-glycosidic bond). This yields free uracil and DNA with an apyrimidinic site. Apyrimidinic endonuclease (AP endonuclease) recognizes this site and cleaves the phosphodiester bond on the 5' side of the deoxyribose moiety. This is followed in bacteria by the nick translation activity of DNA polymerase I, which inserts dTTP as a replacement for the dUMP that was removed, displacing the deoxyribose phosphate residue in the AP site. This is removed by deoxyribose-5'-phosphatase. The resulting nick is sealed by DNA ligase.