L15 DNA Replication, Repair, and Recombination

Why is there an issue in termination for eukaryotic chromosomes?

-the linear nature of the eukaryotic chromosome

-there is insufficient space for a new primer at the 3' end of the lagging strand

-if a primer were added, there is no 3' end to extend after primer removal

-would shorten the chromosome by a primer length

What are telomeres?

Ends of eukaryotic chromosomes

What is telomerase?

An RNA-directed DNA polymerase, or reverse transcriptase

-Riboucleoprotein: both RNA and protein components

-In higher eukaryotes, active only in germline and stem cells

-chromosome shortening associated with aging and cell senescence

-continues until overhang is long enough for normal machinery to add primer and extend

What are G-quadruplex Structures?

-G-rich overhang sequences at telomeres for G-quartet structures

-May bind capping proteins

-Formed in G rich nucleotide sequences

-Hoogsteen hydrogen binding

-square planar structure

-monovalent cation stabilized

-can also modulate gene expression

What are mutations caused by?

metabolic activities or environmental exposures on DNA

What is the natural rate of mutation>

about 1.o mutation per 100,00 genes per generation (germline, higher eukaryotes)

-somatic cell mutations less problematic but can be a cause of cancer

List some environmental and chemical agents that can damage DNA to generate mutations

-UV light

-Ionizing radiation (can generate hydroxyl radical)

-reactive chemicals

-spontaneous purine hydrolysis (20,000 of 6 billion per day)

What is a point mutation?

single base change, single nucleotide polymorphisms (SNPs)

What is a transition?

Base is replaced with the same type of base (Py or Pu)

What is a transversion?

Purine replaces pyrimidine (or vice versa)

Silent mutation

no change in protein sequence

Nonsense mutation

premature stop of protein synthesis

Missense mutation

alters protein sequence

Indels

Insertions or deletions

-generally involve more than one base (1-1000's)

Frameshift mutation

indel in protein coding sequence, if not a multiple of 3

What can nitrous acid do to bases?

Nitrous acid can deaminate bases

-can cause both AT to GC and GC to AT transitions

Are reactive oxygen species a part of normal cell metabolism?

Yes

-superoxide, hydroxy radicals, peroxide

-8-oxoguanin, e.g.

-can yield GC to TA transversion

What does alkylation of purines at N7 position promote?

It promotes hydrolysis of the glycosidic bond

-repair system is error prone

How many purines go through spontaneous hydrolysis per day?

about 20,000 of 6 billion per day

What is the Ames Test?

Ames is a simple test for mutagenesis

-Salmonella typhimurium

-His-

Cannot grow unless media is supplemeted with histedine

-If correct mutation arises, can become his+

-10^9 bacteria plated and wait for 2 days

How many molecular lesions does a typical mammalian cell have to its DNA per day?

About 100,000 molecular lesions

What does the enzyme photolyase do?

Photolyase uses light to exvite the electrons in cyclobutane ring catalyzing the retro-Diels-Alder reaction

-separates adjacent linked pyrimidine rings by "base flipping"

Do placental mammals have photolyase?

No

What does O6-alkylguanine-DNA alkyltransferase do?

-removes methyl group with a Cys residue

-Permanent enzyme methylation

What does DNA glycosylase do?

It cleaves the N-glycoside linkage of a damaged base and the deoxyribose

-8-oxoguanine and uracil in DNA (idk what that means) (hopefully not very important)

What does Uracil-DNA glycolase (UDG) do?

It recognizes U-G mismatch and excises

How are Apurinic or Apyrimidinic sites resolved?

They are resolved through the action of nucleases that remove the residue, DNA polymerase (pol I in bacteria' DNA polymerase b in mammals), and DNA ligase

What is nucleotide Excision Repair?

-it is contained by all cells

-corrects pyrimidine dimers and other bulky lesions that distort the bases from their normal positions

What is the UvrABC nuclease system in E. coli?

-Two cuts are made in the DNA in the damaged strand, one on either side of the damage

-E.coli excision nucleases Uvr A, B, and C

-Excised DNA of 11-12 bases is removed by UcrD helicase

-The gap is filled, and the nick is ligated

What is Xeroderma Pigmentosum?

Disorder where patients are deficient in one of the activities needed for nucleotide excision repair.

-extreme sensitivity to light

What is mismatch repair?

Single-strand repair mechanism that correct helix distorting base mispairings.

-proofreading errors

-replication slippage

-errors that escaped editing functions during transcription

*Key feature is capacity to distinguish between old and newly synthesized strands

In prokaryotes, what marks the template strand?

methylation

In prokaryotes, what recognizes mismatches?

MutS homodimer

...and it recruits additional proteins to selectively cleave out portion of new strand

How does mismatch repair work in Eukaryotes?

Eukaryotes have homologs of MutS and L may use lagging strand status (unsealed nicks) to mark new strand

When do double strand breaks occur?

With interstrand cross-links, topoisomerase inhibition/deficiency, and with ionizing radiation damage

How many dividing cells in culture exhibit a chromosomal break?

5-10% of dividing cells in culture exhibit a chromosomal break

What are the two pathways for double strand break repair?

-Non-homologous end joining (NHEJ)

-Homologous Recombination

What is Non-homologous end joining?

Removes or extends ssDNA and brings two ends together for ligation

-error prone due to no requirement for sequence homology

What protein is at the core of end joining complex?

the protein Ku

iClicker Question:

What DNA anomaly is repaired by mismatch repair?

replication errors

What is DNA Recombination?

Recombination of DNA is the rearrangement of DNA sequences by exchanging segments from different molecules

-Exchange of dsDNA between maternal and paternal chromosomes prior to gamete formation causes linkage disequilibrium in genetics

What are the two main types of recombination?

-General recombination

-Site-specific recombination

What is general recombination?

Occurs between homologous DNA molecules (most common during meiosis)

-homologous recombination

-occurs in all living organisms

What is site-specific recombination?

The exchange of sequences only requires short regions of DNA homology

-observed in transposition variation

-observed in bacteriophage DNA integration in E. coli DNA

What is the Holliday Model of General Recombination?

-Two homologous DNA molecules are paired

-Two of the DNA strands are cleaved, one in each

-The two nicked strand segments cross over, DNA ligase seals cuts to form a Holliday intermediate

-Branch migration, via basepair exchange, leads to transfer of a segment of DNA

-Second series of DNA strand cuts occur on Chi structures

-Can be resolved in 2 ways

-DNA polymerase fills any gaps, and DNA ligase seals cuts

What does RecBCD do?

-contains both nuclease and helicase activity

-binds ends of dsDNA and unwinds

-degrades back to specific sites

-Recruits RecA

What does RecA do?

Mediate Strand Exchange

-ATP dependent

-RecA partially unwinds the duplex

-Exchanges the ssDNA with the corresponding strand on the dsFNA

-3 stranded intermediate

What is RuvA

-2 homotetramers form around Holiday junction

What is RuvB?

-ATPase

-2 hexamers form around dsDNA on opposite

What do RuvA and RuvB do to DNA?

DNA is pulled through the RuvB tings and pushed apart within RuvA

What is RuvC?

Nuclease that resolves junctions

How often do damaged replication forks occur?

Damaged replication forks are commonplace

-at lease once per bacterial cell division

-around 10x per eukaryotic cell cycle

What is thought to be the primary function of homologous recombination?

idk? replication forks??

What does the presence of a nick in the DNA template cause?

It causes a replication fork to collapse

-replisome dissociates

How does repair of the collapsed replication fork begin?

-Repair begins with RecBCD an RecA mediating strand invasion of the newly synthesized 3'end into the homologous dsDNA

-branch migration by RuvAB occurs

-RevC resolves

What does the 5' end of the nick become?

5' end of the nick becomes the 5' end of an Okazaki fragment

-Origin-independent replication restart

-Restart primsome

What is homologous end-joining?

Nonmutagenic alternative to double strand break repair to nonhomologous end-joining

-copies sequences from a homologous chromosome

Why are both dsDNA ends cut back?

They are cut back to yield single-stranded ends

What mediates strand invasion of a 3' end?

Rad51

What does DNA polymerase do in double strand break repair?

DNA polymerase extends invading and noninvading 3' ends

What are BRCA1 and BRCA2?

BRCA1 and BRCA2 are proteins that interact with Rad51

-mutant versions are strongly associated with cancer

What is CRISPR?

Clustered Regularly Interspersed Short Palindromic Repeats

-Arrays of DNA with hundred of repeating palindromic sequences

-20-50 bp long

What are protospacers?

They are interspersed among arrays of FNA with hundreds of repeating palindromic sequences

-protospacers containDNA sequences from bacteriophage DNA

How many base transcripts does transcription of the CRISPR locus generate

~30 base transcripts

CRISPR-Cas for Modifying Genomes

-most common approach to gene knockouts

-can be used to activate specific genes

Give examples of CRISPR-Cas uses for therapeutics

-cystic fibrosis, progeria

-sickle cell therapeutic