DNA REPAIR

What are the main differences between Homologous recombination during meiosis and Homologous recombination?

However, several meiosis-specific proteins direct them to perform their tasks somewhat differently, resulting in the distinctive outcomes observed for meiosis. Another important difference is that, in meiosis, recombination occurs preferentially between maternal and paternal chromosomal homologs rather than between the newly replicated, identical DNA duplexes that pair in double-strand break repair. In the sections that follow, we describe in more detail those aspects of homologous recombination that are especially important for meiosis.

Structure of a portion of the Rad52 protein

This doughnutshaped structure is composed of 11 subunits. Single-strand DNA has been modeled into the deep groove running along the protein surface. Rad52 helps load Rad51 onto single-strand DNA to form the nucleoprotein filament that carries out strand exchange. Rad52 also acts later to re-form the double helix and complete the homologous recombination reaction

What is the least amount of nucleotides required to bind in order to stabilize the invading strand?

(at least 15 nucleotides) stabilizes the invading strand and leads to strand exchange

Homologous Recombination (HR)

A repair pathway for double strand breaks involving the undamaged sister chromatid, which provides a template for high fidelity repair. (BRCA1 and BRCA2 involved in this) Repairing double-strand breaks by homologous recombination is more difficult to accomplish but restores the original DNA sequence.

What is the loss of heterozygosity? When does it happen? Why is it dangerous?

Because maternal and paternal chromosomes differ in DNA sequence at many positions along their lengths, this type of repair can convert the sequence of the repaired DNA from the maternal to the paternal sequence or vice versa. It can have severe consequences if the homolog used for repair contains a deleterious mutation, because the recombination event destroys the "good" copy. Loss of heterozygosity, although rare, is a critical step in the formation of many cancers

Phenotype and process effected of BRCA1

Breast and ovarian cancer Repair by homologous recombination

Phenotype and process effected of BRCA2

Breast, ovarian, and prostate cancer Repair by homologous recombination

Under physiological conditions what is the differentiation of the likelihood of the different dna lesions that can occur over 24 hours.

Depurination happens most often

What is the function of Mre11

FURTHER PROCESSING OF 5′ ENDS BY NUCLEASE also involved in regular homologues recombination

Mre11 complex

Mre11 is a nuclease that processes damaged DNA in preparation for homologous recombination

Describe the proteins responsible for homologous recombination in bacteria and in humans?

RecA in E. coli and Rad51 in all eukaryotes

RuvA

Tetramer Binds DNA, promotes branch migration coordinates this movement, threading the DNA strands to avoid tangling no ATP

How can this nick broken fork be repaired?

The broken fork can be flawlessly repaired (Figure 5-50) using the same basic homologous recombination reactions we discussed above for the repair of double-strand breaks. With slight modifications,

What determines which route it takes?

The cutting of either side?

Rad51

The eukaryotic homolog of RecA. An ATPase that binds single stranded DNA

What is the function of translesion polymerases,

The highly accurate replica - tive DNA polymerases stall when they encounter damaged DNA, and in emer - gencies cells employ versatile, but less accurate, backup polymerases,

Holliday junction or cross-strand exchange

The middle stage of balagan The initially formed structure (A) is usually drawn with two strands crossing, as in Figure 5-54. An isomerization of the Holliday junction (B) produces an open, symmetrical structure that is bound by specialized proteins. (C) These proteins "move" the Holliday junctions by a coordinated set of branchmigration reactions (see Figure 5-57 and Movie 5.8). (D) Structure of the Holliday junction in the open form depicted in (B). The Holliday junction is named for the scientist who first proposed its formation

How is it if both crossovers and non crossovers produce heteroduplex's that we deviate from the mendelian rules of genetics?

We have seen that both crossovers and non-crossovers produce heteroduplex regions of DNA. If the two strands that make up a heteroduplex region do not have identical nucleotide sequences, mismatched base pairs are formed, and these are often repaired by the cell's mismatch repair system (see Figure 5-19). However, the mismatch repair system cannot distinguish between the paternal and maternal strands and will randomly choose the strand to be used as a template. As a consequence, one allele will be lost and the other duplicated (Figure 5-59), resulting in net "conversion" of one allele to the other. Thus, gene conversion, originally regarded as a mysterious deviation from the rules of genetics, can be seen as a straightforward consequence of the mechanisms of homologous recombination this happens on a relatively minor scale as DNA sequence changes at the site of recombination

heteroduplex DNA

a hybrid DNA molecule made by annealing complementary strands from similar, but non-identical DNA molecules; the heteroduplex region is where the sequences are not perfectly complementary

What is the function of glycosylase?

cleaves the altered base

What are the spontaneous alterations that require DNA repair

oxidative damage (red) hydrolytic attack (blue) methylation (green)

How is the importance of DAN repair known?

several percent of the coding capacity of most genomes is devoted solely to DNA repair functions.

Which nucleotide doesnt have a deamination?

thymine

Does RecA need ATP

yes

How do chemical modifications of nucleotides produce full fledged mutations

) Deamination of cytosine, if uncorrected, results in the substitution of one base for another when the DNA is replicated. As shown in Figure 5-38, deamination of cytosine produces uracil. Uracil differs from cytosine in its base-pairing properties and preferentially base-pairs with adenine. The DNA replication machinery therefore adds an adenine when it encounters a uracil on the template strand. (B) Depurination can lead to the loss of a nucleotide pair. When the replication machinery encounters a missing purine on the template strand, it may skip to the next complete nucleotide as illustrated here, thus producing a nucleotide deletion in the newly synthesized strand. Many other types of DNA damage (see Figure 5-37), if left uncorrected, also produce mutations when the DNA is replicated

Double Holliday Junction

An intermediate structure temporarily connecting chromatids of homologous chromosomes that forms during homologous recombination.

How does nucleotide excision repair work with RNA polymerase in bacteria and in humans

In bacteria the Polymerase falls when recognizing the mutation in humans it reverses allows for the fix and the continues

Difference between endonuclease and exonuclease

endo breaks from within exo from without

How are BRCA mutations invloved with homologues recombination?

the Brca1 and Brca2 proteins, were first discovered because mutations in their genes lead to a greatly increased frequency of breast cancer. Because these mutations cause inefficient repair by homologous recombination, accumulation of DNA damage can, in a small proportion of cells, give rise to a cancer r. Brca1 regulates an early step in broken-end processing; without it, such ends are not processed correctly for homologous recombination and instead are repaired inaccurately by the nonhomologous end-joining pathway Brca2 binds to the Rad51 protein, preventing its polymerization on DNA, and thereby maintaining it in an inactive form until it is needed. Normally, upon DNA damage, Brca2 helps to bring Rad51 protein rapidly to sites of damage and, once in place, to release it in its active form onto single-strand DNA.

branch migration

the lateral movement of a Holliday junction DNA is spooled through the Holliday junction by continually breaking and re-forming base pairs

Phenotype and process effected of of MSH2, 3, 6, MLH1, PMS2

-colon cancer -mismatch repair

Why is AP endonuclease called as such?

. AP endonuclease is so-named because it recognizes any site in the DNA helix that contains a deoxyribose sugar with a missing base; such sites can arise either by the loss of a purine (apurinic sites) or by the loss of a pyrimidine (apyrimidinic sites)

What are the possible results of Homologous recombination during meiosis

1) closely resembles the double-strand break repair reaction shown in Figure 5-48 and results in chromosomes that have been "repaired" but have not crossed over 2)The other (left side with strand breaks as shown by the blue arrows) proceeds through a double Holliday junction and produces two chromosomes that have crossed over. During meiosis, homologous recombination takes place between maternal and paternal chromosome homologs when they are held tightly together

Describe regulation of homologous recombination

Cells go to great lengths to minimize the risk of mishaps of these types; indeed, nearly every step of homologous recombination is carefully regulated. For example, the first step, processing of the broken ends, is coordinated with the cell cycle: the nuclease enzymes that carry out this process are activated (in part, by phosphorylation) only in the S and G2 phases of the cell cycle, when a daughter duplex (either as a partially replicated chromosome or a fully replicated sister chromatid) can serve as a template for repair (see Figure 5-50). The close proximity of the two daughter chromosomes disfavors the use of other genome sequences in the repair process. The loading of RecA or Rad52 onto the processed DNA ends and the subsequent strand-exchange reaction are also tightly controlled. Although these proteins alone can carry out these steps in vitro, a series of accessory proteins, including Rad52, is needed in eukaryotic cells to ensure that homologous recombination is efficient and accurate (Figure 5-51). There are many such accessory proteins, and exactly how they coordinate and control homologous recombination remains a mystery. We do know that the enzymes that catalyze recombinational repair are made at relatively high levels in eukaryotes and are dispersed throughout the nucleus in an inactive form. In response to DNA damage, they rapidly converge on the sites of DNA damage, become activated, and form "repair factories" where many lesions are apparently brought together and repaired

What disease is ascociated with a problem in this pathway of Coupling of nucleotide excision repair to transcription?

Cockayne syndrome

Phenotype and process effected of Fanconi anemia groups A-G

Congenital abnormalities, leukemia, genome instability DNA interstrand cross-link repair

pyrimidine dimer formation

Covalent bonds between 2 T's Mainly Thymine Dimers Distort helix and inhibit replication Xeroderma pigmentosum--> repair mech. defective, tumors on skin surface

DNA hybridization

DNA double helices can re-form from their separated strands in a reaction that depends on the random collision of two complementary DNA strands. The vast majority of such collisions are not productive, as shown on the left, but a few result in a short region where complementary base pairs have formed (helix nucleation). A rapid zippering then leads to the formation of a complete double helix. Through this trial-and-error process, a DNA strand will find its complementary partner even in the midst of millions of nonmatching DNA strand

How does translesion polymerase differ from the replicative polymerase?

Human cells have seven translesion polymerases, some of which can recog - nize a specific type of DNA damage and correctly add the nucleotide required to restore the initial sequence. Others make only "good guesses," especially when the template base has been extensively damaged. These enzymes are not as accurate as the normal replicative polymerases when they copy a normal DNA sequence. For one thing, the translesion polymerases lack exonucleolytic proofreading activity; in addition, many are much less discriminating than the replicative polymerase in choosing which nucleotide to incorporate initially. Presumably for this reason, each such translesion polymerase is given a chance to add only one or a few nucleotides before the highly accurate replicative polymerase resumes DNA synthesis.

Describe how Experiment demonstrating the rapid localization of repair proteins to DNA double-strand breaks

Human fibroblasts were x-irradiated to produce DNA double-strand breaks. Before the x-rays struck the cells, they were passed through a microscopic grid with x-ray-absorbing "bars" spaced 1 μm apart. This produced a striped pattern of DNA damage, allowing a comparison of damaged and undamaged DNA in the same nucleus. (A) Total DNA in a fibroblast nucleus stained with the dye DAPI. (B) Sites of new DNA synthesis due to repair of DNA damage, indicated by incorporation of BudR (a thymidine analog) and subsequent staining with fluorescently labeled antibodies to BudR (green)

Phenotype and process effected of 46 BR patient

Hypersensitivity to DNA-damaging agents, genome instability DNA ligase I

Hypoxanthine

Hypoxanthine is the simplest purine base capable of pairing specifically with C, but hypoxanthine is the direct deamination product of A

Describe the enzyme-catalyzed branch movement at a holliday junction

In E. coli, a tetramer of the RuvA protein (green) and two hexamers of the RuvB protein (yellow) bind to the open form of the junction. The RuvB protein, which resembles the hexameric helicases used in DNA replication, uses the energy of ATP hydrolysis to spool DNA rapidly through the Holliday junction, extending the heteroduplex region . The RuvA protein coordinates this movement, threading the DNA strands to avoid tangling.

How does Nucleotide excision repair work in bacteria?

In bacteria, after a multienzyme complex has recognized a lesion such as a pyrimidine dimer (see Figure 5-39), one cut is made on each side of the lesion, and an associated DNA helicase then removes the entire portion of the damaged strand. The excision repair machinery in bacteria leaves the gap of 12 nucleotides shown

How does Nucleotide excision repair work in humans?

In humans, once the damaged DNA is recognized, a helicase is recruited to unwind the DNA duplex locally. Next, the excision nuclease enters and cleaves on either side of the damage, leaving a gap of about 30 nucleotides. The nucleotide excision repair machinery in both bacteria and humans can recognize and repair many different types of DNA damage

How does Nucleotide excision repair work?

In this pathway, a large multienzyme complex scans the DNA for a distortion in the double helix, rather than for a specific base change. Once it finds a lesion, it cleaves the phosphodiester backbone of the abnormal strand on both sides of the distortion, and a DNA helicase peels away the single-strand oligonucleotide containing the lesion. The large gap produced in the DNA helix is then repaired by DNA polymerase and DNA ligase

How is homologous recombination usually brought about?

Instead, as we shall see, homologous recombination is brought about through a carefully controlled set of reactions that allow two DNA duplexes to sample each other's sequences without fully dissociating into single strand

What causes serious damage in the form of "double strand breakage"?

Ionizing radiation, replication errors, oxidizing agents, and other metabolites produced in the cell cause breaks of this type.

When does Homologous Recombination (HR) usually take place

It typically takes place after the DNA has been duplicated (when a duplex template is available) but before the cell has divided (in S and G2 phases),

Phenotype and process effected of Ataxia telangiectasia (AT)

Leukemia, lymphoma, γ-ray sensitivity, genome instability instability ATM protein, a protein kinase activated by double-strand breaks

How does Spo11 resemble topoisomerase

Like a topoisomerase, Spo11, after catalyzing this reaction, remains covalently bound to the broken DNA

What happens in depurination

N-glycosyl linkages to deoxyribose hydrolyze, a spontaneous reaction

What is The most common type of thymine dimer how does it occur?

Occurs through exposure to UV radiation

Phenotype and process effected of Werner syndrome

Premature aging, cancer at several sites, genome instability Accessory 3ʹ-exonuclease and DNA helicase used in repair

The stages of meiosis in the correct sequence

Prophase I, Metaphase I, Anaphase I, Telophase I, Prophase II, Metaphase II, Anaphase II, Telophase II

How dos RecA work? When does it leave?

RecA first binds cooperatively to the invading single strand, forming a protein-DNA filament that forces the DNA into an unusual configuration: groups of three consecutive nucleotides are held as though they were in a conventional DNA double helix but, between adjacent triplets, the DNA backbone is untwisted and stretched out This unusual protein-DNA filament then binds to duplex DNA in a way that stretches the duplex, destabilizing it and making it easy to pull the strands apart RecA hydrolyzes ATP, and the steps described above require that each RecA monomer along the filament be in the ATP-bound state. However, the searching itself does not require ATP hydrolysis; instead, the process occurs by simple molecular collision, allowing many potential sequences to be rapidly sampled. Once the strand-exchange reaction is completed, however, ATP hydrolysis is necessary to disassemble RecA from the complex of DNA molecules. At this point, repair DNA polymerases and DNA ligase can complete the repair process, as shown in Figure 5-48

Phenotype and process effected of Xeroderma pigmentosum (XP) groups A-G

Skin cancer, UV sensitivity, neurological abnormalities Nucleotide excision repair

ATM protein

The ATM protein is a large kinase needed to generate the intracellular signals that sound the alarm in response to many types of spontaneous DNA damage (see Figure 17-62), and individuals with defects in this protein therefore suffer from the effects of unrepaired DNA lesions.

In V(D)J joining what process is employed to break the DNA and then put it back together? How does this work? What protein is responsible.

The Ku protein, a heterodimer that grasps the broken chromosome ends. The additional proteins shown are needed to hold the broken ends together while they are processed and eventually joined covalently

base excision repair 4 stages

This pathway starts with a DNA glycosylase. Here, the enzyme uracil DNA glycosylase removes an accidentally deaminated cytosine in DNA. After the action of this glycosylase (or another DNA glycosylase that recognizes a different kind of damage), the sugar phosphate with the missing base is cut out by the sequential action of AP endonuclease and a phosphodiesterase. (These same enzymes begin the repair of depurinated sites directly.) The gap of a single nucleotide is then filled by DNA polymerase (low processivity) and DNA ligase. The net result is that the U that was created by accidental deamination is restored to a C.

What is done for the 5-methyl-cytosine mutation

To help in repairing deaminated methylated C nucleotides, a special DNA glycosylase recognizes a mismatched base pair involving T in the sequence T-G and removes the T. This DNA repair mechanism must be relatively ineffective, however, because methylated C nucle - otides are exceptionally common sites for mutations in vertebrate DNA. It is striking that, even though only about 3% of the C nucleotides in human DNA are methylated, mutations in these methylated nucleotides account for about one-third of the single-base mutations that have been observed in inherited human diseases

Phenotype and process effected of XP variant

UV sensitivity, skin cancer Translesion synthesis by DNA polymerase ν

Phenotype and process effected of Cockayne syndrome

UV sensitivity; developmental abnormalities Coupling of nucleotide excision repair to transcription

When will the cell purposefully break the double strand?

V(D)J joining, a specific recombination process through which antibody and T cell receptor diversity is generated in developing B and T cells

What can cause a replication fork to break

a single-strand nick or gap in the parental DNA helix just ahead of a replication fork. When the fork reaches this lesion, it falls apart—resulting in one broken and one intact daughter chromosome

What is Spo11?

a specialized protein (its called Spo11 in budding yeast) breaks both strands of the DNA double helix in one of the recombining chromosomes It is necessary in order to use homologous recombination where there is no break in the DNA originally

What is the disease called for those who have a problem with the ATM protein? What are the symptomes?

ataxia telangiectasia (AT), the symptoms of which include neurodegeneration, a predisposition to cancer, and genome instability.

two of the most common pathways of DNA correction

base excision repair NUCLEOTIDE EXCISION REPAIR

Why is DNA suitable for repair?

because it carries two separate copies of all the genetic information—one in each of its two strands. Thus, when one strand is damaged, the complementary strand retains an intact copy of the same information, and this copy is generally used to restore the correct nucleotide sequences to the damaged strand.

How can deamination explain why we use DNA as opposed to RNA

because the body might get confused with Uracil in correction

Why even in the outcome of RELEASE OF INVADING STRAND might we find mismatch?

because the template that was taken to copy comes from the other parental chromosome

nonhomologous end joining what is it and why is it good enough ? where is it good enough?

broken ends are simply brought together and rejoined by DNA ligation, generally with the loss of nucleotides at the site of joining (Figure 5-46). This end-joining mechanism, which can be seen as a "quick and dirty" solution to the repair of double-strand breaks, is common in mammalian somatic cells. Although a change in the DNA sequence (a mutation) results at the site of breakage, so little of the mammalian genome is essential for life that this mechanism is apparently an acceptable solution to the problem of rejoining broken chromosomes. B

How do cells direct DNA repair to the DNA sequences that are most urgently needed.

by linking RNA polymerase, the enzyme that transcribes DNA into RNA as the first step in gene expression, to the nucleotide excision repair pathway

Phenotype and process effected of Bloom syndrome

cancer at several sites, stunted growth, genome instability DNA helicase needed for recombination

What are especially notories mutation cites in vertabrates

deamination of 5-methyl-cytosine

What is An alternative to base and nucleotide excision repair processes? When is it used?

direct chemical reversal of DNA damage, and this strategy is selectively employed for the rapid removal of certain highly mutagenic or cytotoxic lesions. For example, the alkylation lesion O6-methylguanine has its methyl group removed by direct transfer to a cysteine residue in the repair protein itself, which is destroyed in the reaction

crossover control

each pair of chromosomes only acquires 1-2 crossovers, but in most cases at least one event occurs per chromosome. regulatory mechanism ensures the roughly even distribution of crossover points along chromosomes ensures that each chromosome— no matter how small—undergoes at least one crossover every meiosis.

What should be the distribution of genes, what is the variation of this called and why does it happen?

each parent makes an equal genetic contribution to an offspring. One complete set of nuclear genes is inherited from the father and one complete set is inherited from the mother. Underlying this law is the accurate parceling out of chromosomes to the germ cells (eggs and sperm) that takes place during meiosis. Thus, when a diploid cell in a parent undergoes meiosis to produce four haploid germ cells, exactly half of the genes distributed among these four cells should be maternal (genes inherited from the mother of this parent) and the other half paternal divergence from their expected distribution during meiosis that is known as gene conversion and it is caused by mis match correction . Such repair can "erase" nucleotide sequences on either the paternal or the maternal strand. The consequence of this mismatch repair is gene conversion, detected as a deviation from the segregation of equal copies of maternal and paternal alleles that normally occurs in meiosis.

Why might be a reason for CG islands being less present in coding regions

higher liklihood of deamination of 5-methyl-cytosine

How does DNA Damage Delay Progression of the Cell Cycle

in mammalian cells, the presence of DNA damage can block entry from G1 into S phase, it can slow S phase once it has begun, and it can block the transition from G2 phase to M phase. These delays facilitate DNA repair by providing the time needed for the repair to reach completion.

Where else do we find homologous recombination?

in meiosis

What is Nucleotide excision repair usually meant for

pyrimidine dimers usually but others as well

how many double helix's do we have during meiosis

really 4 doubles meaning 8 strands but if we look closely at recombination in will be 4 strands

What is the sliding clamp's role in serious DNA damage recognition

replicative polymerase stalles at a site of DNA damage is recognized by the cell as needing rescue. Specialized enzymes covalently modify the sliding clamp (typically, it is ubiquitylated which releases the replicative DNA polymerase and, together with damaged DNA, attracts a translesion polymerase specific to that type of damage. Once the damaged DNA is bypassed, the covalent modification of the clamp is removed, the translesion polymerase dissociates, and the replicative polymerase is brought back into play

What happens in DEAMINATION

spontaneous deamination of cytosine turns into uracil

. Meiosis

the process by which a diploid cell gives rise to four haploid germ cells, Meiosis produces germ cells in which the paternal and maternal genetic information (red and blue) has been reassorted through chromosome crossovers. In addition, many short regions of gene conversion occur,

Ruv B

two hexamers bind to the open form of the junction. resemblee hexameric helicases used in DNA replication uses the energy of ATP hydrolysis to spool DNA rapidly through the Holliday junction, extending the heteroduplex region as shown

In what type of nucleotide will we most likely find dimer connections

two neighboring pyrimidine bases C or T