DNA Repair and Recombination

Question 1

Distinguish between the types of “small” mutations (deletions, insertions, altered)

Answer 1

Deletions: When a base or chunk of DNA is deleted

Insertions: when DNA is added

Altered: base pairs are altered or changed to something else

Commonly result in SNPs or single nucleotide polymorphisms

Question 2

Missense

Answer 2

Conversion of one base pair to another that results in different codon for another amino acid

Question 3

Nonsense

Answer 3

A stop codon is created

Question 4

Spice Variants

Answer 4

Get a mutation in the splice site which alters splicing pattern

Question 5

Base Substitutions/modification

Answer 5

A type of “small” scale mutations that involves the replacement or modification of a single base

Get transitions (Pyrimidine is replaced with a pyrimidine or purine for purine) or transversions (Pu for Py)

Can also involve methylations or unnatural bases

Question 6

Deletions

Answer 6

Another small scale mutation in which one or more nucleotides are eliminated from a sequence. Now have a frameshift mutation

Question 7

Insertions

Answer 7

Another type of small scale mutations in there are copy/duplicative transpositions (duplicated copy moved to another location) or non-copy transposition (movement to a new location-rare in humans). These also result in frameshifts

Question 8

Large Scale Changes

Answer 8

These are on the chromosomal level and still caused by deletions, duplications, inversions, translocation fusions. Examples of translocations include the BCR-ABL oncogene and various MLL gene fusions in leukemia. Mechanisms include amplifications (gene duplications), deletions, translocations, and loss of heterozygosity.

Question 9

De novo mutations

Answer 9

The can occur in both somatic cells as well as germ line cells. Sources of de novo mutations include chemical attacks (depurination (loss of A or G) or deamination (C to U)), environmental exposure and copying errors. These require repair using one or more repair mechanisms. Mutations associated with disease are usually de novo.

Question 10

Somatic mutations

Answer 10

They are not heritable and affect only those cells that result from mitotic division. Mutations can accumulate in these cells, giving rise to diseases such as cancer.

Question 11

Germline mutations

Answer 11

are heritable. They affect the whole individual (all cells) thus increasing the susceptibility to diseases that arise from secondary mutations.

Question 12

Single Nucleotide Polymorphisms (SNPs)

Answer 12

Polymorphisms arising with a frequency >0.01. Greater than 1 in 100, it is a SNP.

Question 13

Deamination

Answer 13

It leads to the conversion of a C to a U. This will then cause the G that was complementary to the C to be replaced by an A during DNA replication on one strand but the U will be replaced by a C on the other. Now have a mutation in one cell that will get that daughter strand

Question 14

Depurination

Answer 14

It leads to the loss of an A or a G which leads to a gap that needs to be fixed. If it is not fixed, the one strand will be copied and now be missing a base where the other strand will be normal. One cell will be mutated.

Question 15

3 Consequences of not fixing a mutation

Answer 15

1) (transient) Cell-cycle arrest: There are checkpoints that block you from proceeding through the cell cycle if you’re damaged.
2) Apoptosis: Results in inhibition of transcription, replication, and chromosome segregation to result in cell death
3) Cancer, ageing, inborne disease: consequences that result if the error is not fixed or removed and cell is not killed.

Question 16

Describe the causes of various DNA lesions

Answer 16

UV radiation, environmental attacks, x-rays, oxygen radicals, chemotherapy reagents. These are environmental attacks. Also get errors that naturally occur during synthesis, as well as deamination and depurination

Question 17

What are the 3 major classes of DNA repair mechanisms?

Answer 17

1) Direct repair: damage reversal. Uses MGMT to remove a methylation for example
2) Excision Repair: Removal and replacement. Types inclue base excision repair (BER), nucleotide excision repair (NER), transcription coupled repair (TCR), and Mismatch Repair (MMR)
3) Double-strand Break Repair: Uses homologous End Joining (HEJ) and Non-Homologous End Joining (NHEJ)

Most DNA lesions can be repaired by more than one mechanism

Question 18

Direct Repair Mechanism

Answer 18

An MGMT protein with an -SH group can bind to a methyl group on the guanine that inhibits it from H-bonding to cytosine to remove it. It is the most energetically costly.

Question 19

Nucleotide Excision Repair (NER)

Answer 19

An enzyme complex recognizes non-specific bulky lesions. Almost any lesion can be repaired by global NER except DSBs. A nuclease then cleaves on either side of the lesion distant from the damage. Helicase removes the DNA and Polymerase fills the gap with ligase joining the ends. IT IS REMOVING A CHUNK OF DNA, NOT JUST ONE BASE

Question 20

Base Excision Repair (BER)

Answer 20

It is the most common repair mechanism. It is using a scanning, “flip-out” method to check the bases. Specific glycosylases recognize specific altered bases and generate AP (apurinic or apyrimidinic) sites. AP endonucleases and phosphodieterases cut the sugar backbone, remove the base then the gap is filled and sealed.

Question 21

Transcription Coupled Repair (TCR)

Answer 21

It is associated with ACTIVELY TRANSCRIBED DNA. RNA polymerase stalls and backs up at the damage site. Elongation factors function in repair. Several XP proteins are associated with the TFIIH which is the natural helicase that identifies lesions on actively transcribed DNA. The stalling of polymerase recruits these proteins to repair the DNA. TFIIH is recruited and then recruits XPG and XPF endonucleases which cleave around the damage. Cockayne syndrome and Xeroderma pigmentoma are caused by mutations in TCR.

Question 22

Mismatch Repair (MMR)

Answer 22

It is used to correct errors FOLLOWING replication. It is a backup mechanism. There is an error in which two bases can’t correctly base pair. MutS scans the DNA and recognizes this damage. Then MutL will look for a nick in the DNA that ligase hasn’t joined yet, bind, loop back to the damage and cleave away the strand. Polymerase is then recruited again to fill in the gap.

Question 23

Homologous End Joining (HEJ)

Answer 23

A DNA repair mechanism that attempts to repair double stranded breaks (DSBs). It is active ONLY in the S-phase and G2-phase because it needs a homologous complement to repair off of. In this case it uses the newly synthesized sister chromatid. A double strand break occurs, the nuclease chews back to get 3’-overhangs, one then aligns with the sister chromatid and polymerase synthesizes the strand accordingly. Now a template is provided for the other strand. RecA/Rad51 proteins catalyze strand invasion and the formation of the triple-stranded intermediates. You don’t get an exchange of genetic information because it is an exact copy! This is also seen in Homologous Recombination.

Question 24

Non-Homologous End Joining (NHEJ)

Answer 24

A DNA repair mechanism that is attempting to repair DSBs. Found in Go/G1 phase because there is no longer a sister chromatid available to be used as a template. It is mutagenic because double strands are degraded and not replaced. It is an emergency solution. You get a double stranded break, then Ku heterodimers recognize the broken ends. It recruits additional proteins, ends are processed and ligation occurs.

Question 25

Describe the major steps of general recombination

Answer 25

1) DSB occurs
2) Sister chromatids pair (have this because they replicated)
3) Degradation of ends to form 3’ overhangs
4) Strand invasion guided by RecA/Rad51
5) Branch migration to its complement and DNA synthesis
6) DNA helix is reformed
7) DNA ligation

NO EXCHANGE OF GENETIC INFO BECAUSE ITS AN EXACT COPY

Question 26

Strand Invasion

Answer 26

When one strand invades the strand of the newly replicated “template” forming a triple-stranded intermediate.

Question 27

Homologous Chromosome

Answer 27

Chromosome that is the same “chromosome number” with the same alleles but may have different genes within the alleles.

Question 28

Sister Chromatid

Answer 28

exact copies of one another and thus contain the same genetic information

Question 29

Crossover

Answer 29

Where the two strands of DNA cross over and exchange information. See a lot more crossing over in highly repetitive elements.

Question 30

Heteroduplex joint

Answer 30

It is where strands from two different DNA helices have base-paired. Where, on the DNA level, there is genetic information from each of the chromosomes. It is no the original pairing. Heterduplexes can occur where the holliday junctions were resolved as noncross-overs or a cross-over event.

Question 31

Holliday Junction

Answer 31

It is where two strands have crossed over. Over 90% of these are resolved without crossing over, meaning they are repaired via NHEJ. How the holliday junction is resolved depends on whether or not there is crossing over.

Question 32

meiotic prophase

Answer 32

Crossing over occurs at meiotic prophase I. You get pairing of homologous chromosomes here. There are meiosis specific enzymes that create DSBs.

Question 33

How does genetic reassortment occur in meiosis?

Answer 33

Via independent reassortment or the ability for the chromosomes to align and separate in any fashion AND recombination by exchanging genetic information

Question 34

Gene Conversion

Answer 34

It is the apparent nonreciprocal exchange of genetic information. Gene conversion during meiosis contributes to variation from mendelian inheritance patterns. Gene conversion involves mismatch repair because if you received genetic info from a homologous chromosome, some of the bases may not longer line up. Thus, mismatch repair takes place to correct this issue.

Question 35

Loss of Heterozygosity

Answer 35

When a heterozygote undergoes recombination, the two chromosomes undergo a crossover event, making two homozygous chromosomes. If the disease is a recessive one, this can now cause cancer is some cells that are now homozygous.