CMB Chapter 6 DNA Replication, Repair, Recombination

Who were Meselson and Stahl?

(1958) showed that DNA replication is semi-conservative - grew cells in heavy nitrogen - extracted DNA and spun in centrifuge tubes with special liquids - is DNA replication conservative, dispersive, or semi-conservative? ruled out conservative and dispersive with experiments

T/F: A bacterial replication fork is asymmetrical because it contains 2 DNA polymerase molecules that are structurally distinct

*FALSE*: identical DNA polymerase molecules catalyze DNA synthesis on the leading and lagging strands; the replication fork is asymmetrical because the lagging strand is synthesized in pieces that are then stitched together

T/F: Okazaki fragments are removed by a nuclease that degrades RNA

*FALSE*: only the RNA primers are removed by an RNA nuclease; Okazaki fragments are pieces of newly synthesized DNA on the lagging strand that are eventually joined together by DNA ligase

T/F: The error rate of DNA replication is reduced both by proofreading by DNA polymerase and by DNA mismatch repair

*TRUE*

T/F: None of the aberrant bases formed by deamination occur naturally in DNA

*TRUE*: if a damaged nucleotide also occurred naturally in DNA, the repair enzyme would have no way of identifying the damage - it would therefore have only a 50% chance of fixing the right strand

T/F: In the absence of DNA repair, genes are unstable

*TRUE*: mutations would accumulate rapidly, inactivating many genes

T/F: Cancer can result from the accumulation of mutations in somatic cells

*TRUE*: usually multiple mutations of specific types need to accumulate in a somatic cell lineage to produce a cancer

What repairs can occur during replication?

- "proofreading/editing" mechanism built into DNA pol III (or α) - recognition of mismatched base pairs - *exonuclease* activity (can cut off an enzyme from a chain) -- polymerase works 5' to 3', but exonuclease works 3' to 5' when something goes wrong it goes back and "deletes" it

Post-replication repair involves 3 processes...

- *excision* (of damaged region of DNA) - *DNA synthesis* (polymerase makes new top strand with bottom strand as template) - *ligase* (repairs break in strand)

What is the process of opening and unwinding DNA helix? (see drawing in notes!)

- *helicase*: separates strands by breaking H bonds, untwists the helix - *primase*: makes an RNA primer at 3' end (only works 5' to 3') - *SSBP (single strand binding proteins)*: bind to each strand and prevent the strands from zipping back together - *DNA polymerase (pol)*: continues to elongate chain (from primase's RNA primer) - 5' to 3' only - *leading/continuous strand*: made in 1 piece by DNA polymerase - *lagging/discontinuous strand*: made in fragments called *Okasaki fragments* -- *exonuclease*: removes RNA nucleotides to remove RNA primers from lagging strand, and DNA polymerase fills in the gaps with DNA nucleotides -- *ligase*: makes phosphodiester bond to combine Okasaki fragments - *toipoisomerase*: works to help unwind DNA strand ahead of *replication fork* - snips 1 phosphodiester bond in each strand of DNA periodically as replication is occurring

Normally, DNA replication has a high fidelity, which means _____

- a low error level (~1 error per 10^9 base pairs) - however, during actual replication process errors are made more often - overall, fidelity depends on proofreading and correcting newly polymerized DNA

What is site-specific integration?

- another type of recombination (besides homologous recombination) - doesn't require homologous sequences (can't be used for repair) - uses specific enzyme (integrase) - recognizes specific nucleotide sequence - occurs in prokaryotes and eukaryotes

What are some examples of errors that occur during replication?

- deamination - depurination - tautomers

What are telomeres?

- end regions of eukaryotic chromosomes, protective caps that prevent shortening of chromosomes as replication occurs - maintained by *telomerase*: can synthesize new telomere sequences (has RNA with it that acts as a template used to elongate fragments) - don't function as well as organism ages - repetitive sequence seen with these

Why is DNA repair necessary?

- fidelity (accuracy) of replication isn't perfect, and errors in replication are 1 source of mutations ex. The cat ate the rat. (correct nucleotide sequence) The c*u*t ate the rat. (base substitution) The cta tet her at. (base deletion)

What is homologous/genetic recombination?

- in addition to mutations, DNA sequences can be changed due to exchanges of segments of DNA through this process - ex. "crossing over" between homologous chromosomes during meiosis - gene exchange occurs between 2 ds (double stranded) DNA molecules - it can occur during... -- integration of plasmids in bacteria -- repair of some ds breaks in DNA -- "crossing over" in meiosis

What is a replication bubble?

- is formed because replication doesn't start at end of DNA strands -- starts in the middle! called *origins of replication* -- prokaryotes have 1 at a time, while eukaryotes have many - replication is bi-directional

What if a double-stranded error occurs?

- more serious - broken ends processed/cut off by nuclease - ends are joined together by DNA ligation - changes the DNA sequence - not ideal, but better than not being repaired at all

Explain why telomeres and telomerase are needed for replication of eukaryotic chromosomes but not for replication of a circular bacterial chromosome

Because DNA polymerase requires a 3'-OH to synthesize DNA, without telomeres and telomerase, the ends of linear chromosomes would shrink during each round of DNA replication. For bacterial chromosomes, which have no ends, the problem does not arise; there will always be a 3'-OH group available to prime the DNA polymerase that replaces the RNA primer with DNA. Telomeres and telomerase prevent the shrinking of chromosomes because they extend the 3' end of a DNA strand. This extension of the lagging strand template provides the "space" to begin the final Okazaki fragments.

What is another source of mutations, besides errors in replication?

DNA damage - chemical or radiation

The lagging strand is synthesized discontinuously at the replication fork because: the lagging strand template is discontinuous. DNA polymerase always falls off the template DNA every ten nucleotides or so. DNA polymerase can polymerize nucleotides only in the 5'-to-3' direction. DNA polymerase removes the last few nucleotides synthesized whenever it stops. None of the above.

DNA polymerase can polymerize nucleotides only in the 5'-to-3' direction.

Describe the consequences that would arise if a eukaryotic chromosome... A. contained only one origin of replication at the exact center of the chromosome B. contained only one origin of replication at one end of the chromosome C. lacked one or both telomeres D. had no centromere ** Assume that the chromosome is 150 million nucleotide pairs in length, a typical size for an animal chromosome, and that DNA replication in animal cells proceeds at about 100 nucleotides per second

Describe the consequences that would arise if a eukaryotic chromosome... *A. contained only one origin of replication at the exact center of the chromosome* it would take more than 8 days to replicate the DNA; the rate of replication would severely limit the rate of cell division *B. contained only one origin of replication at one end of the chromosome* the time required to replicate the chromosome would be double that in A *C. lacked one or both telomeres* a chromosome end that is not "capped" with a telomere would lose nucleotides during each round of DNA replication and would gradually shrink; eventually, essential genes would be lost, and the chromosome's ends might be recognized by the DNA damage-response mechanisms, which would stop cell division or induce cell death *D. had no centromere* centromeres attach mitotic chromosomes to the mitotic spindle - without them, the 2 new chromosomes that result from chromosome duplication would not be partitioned accurately between the 2 daughter cells; therefore, many daughter cells would die without a full set of chromosomes

DNA mismatch repair enzymes preferentially repair bases on the newly synthesized DNA strand, using the old DNA strand as a template. If mismatches were simply repaired without regard for which strand served as template, would this reduce replication errors?

If the old strand were "repaired" using the new strand that contains a replication error as the template, then the error would become a permanent mutation in the genome. The old info would be erased in the process. Therefore, if repair enzymes did not distinguish between the 2 strands, there would be only a 50% chance that any given replication error would be corrected.

A pregnant mouse is exposed to high levels of a chemical. Many of the mice in her litter are deformed, but when they are interbred with each other, all their offspring are normal. Which TWO of the following statements could explain these results? In the deformed mice, somatic cells but not germ cells were mutated. The original mouse's germ cells were mutated. In the deformed mice, germ cells but no somatic cells were mutated. The toxic chemical affects development but is not mutagenic.

In the deformed mice, somatic cells but not germ cells were mutated. The toxic chemical affects development but is not mutagenic.

is DNA replication conservative, dispersive, or semi-conservative?

Meselson and Stahl found that DNA replication is semi-conservative

Which of the following DNA repair processes can occur only immediately after the DNA has been replicated? Repair of deamination. repair of depurination. Mismatch repair. Repair of pyrimidine dimers.

Mismatch repair.

What would be one of the consequences if a eucaryotic chromosome lacked telomeres? DNA replication could not be initiated efficiently. The chromosome would not attach to the mitotic spindle. The chromosome would not be replicated completely. The DNA would not become condensed. The DNA would not be transcribed.

The chromosome would not be replicated completely.

Would you still need telomeres and telomerase to complete eukaryotic chromosome replication if primase always laid down the RNA primer at the very 3' end of the template for the lagging strand?

YES Telomeres and telomerase are still needed even if the last fragment of the lagging strand were initiated by primase at the very 3' end of chromosomal DNA, inasmuch as the RNA primer must be removed.

What is a sliding clamp?

a protein that wraps around DNA strand, helps to hold enzymes onto strand and make replication more efficient

What is a replicon?

a unit of the chromosome where replication occurs - in eukaryotic chromosomes, replication starts at many origins of replication at certain times, non-random

What does DNA polymerase do?

catalyzes chain elongation in 5' --> 3' direction

_______ are fundamental to the function of DNA as genetic material

complementarity and base pairing

Post replication repair - mismatch proofreading

does it matter which strand (after replication) is repaired? yes! -- the newly synthesized strand (because the template strand had the error) - newly synthesized strand must be recognized by nicks in the backbones (where ligase hasn't merged the okasaki fragments yet) - cell recognizes nicks and uses that new strand as the one to be repaired - excises damaged stretch of nucleotides, adds more with DNA polymerase, seals with ligase

DNA replication is considered semiconservative because: after many rounds of DNA replication, the original DNA double helix is still intact. each daughter DNA molecule consists of two new strands which are copied from the parent DNA molecule. each daughter DNA molecule consists of one strand from the parent DNA molecule and one new strand. new DNA strands must be copied from a DNA template.

each daughter DNA molecule consists of one strand from the parent DNA molecule and one new strand.

histones have a lot of _____ charge...

histones have a lot of *positive* charge (and alkaline?), and DNA has a lot of *negative* charge, allowing them to be attracted to each other

Which type of genetic recombination results in an exact exchange of genetic information? site-specific recombination homologous recombination bacterial transformation repair of thymidine dimers

homologous recombination

RNA can be used as a primer for DNA replication in cells because: primase is not found at the replication fork. RNA does not base-pair to the DNA template. primase synthesizes RNA in the 3'-to-5' direction. RNA spontaneously hydrolyzes after DNA polymerization has been started. primase can join ribonucleotides together on a single-stranded DNA template without the need for its own primer.

primase can join ribonucleotides together on a single-stranded DNA template without the need for its own primer.

supercoiling/super helix

when DNA helix is too twisted, caused by too much physical strain ahead of the replication fork