ib 204 - exam 1

Origin - we don’t know how this melts in eukaryotes

what is labeled at A?

what is labeled at B?origin

what is labeled at B?

what

what is labeled at G?

Eukaryotic chromosomes have….

Multiple origins of replication, distributed across the length of a chromosome; end replication bc of losing small piece of dna every round of replication (primer)

Which of the following statements are true in relation to prokaryotic DNA replication:

I. DNA is melted by an initiator protein.

II. Helicase needs to be activated after binding to exposed DNA.

III. Occurs during S-phase of the cell cycle

I is true

Sustained cell division is one of the six hallmarks of cancer, given your understanding predict which of the following contribute to this by stimulating DNA replication:

I. A mutation which impairs binding of ORC to origins.

II. A mutation which increases the amount of MCM2-7 in cells.

III. A mutation which increases phosphorylation of ORC.

II

Apply your understanding of prokaryotic DNA replication to determine which of the following genetic regulatory mechanisms is primarily used by the initiator protein during replication?

protein + DNA interaction

What is the impact of phosphorylation on the ORC?

It releases ORC from the origin and prevents it binding again

Apply your knowledge of eukaryotic DNA replication to state at which stage of the cell cycle MCM2-7 helicase will be unphosphorylated?

G1 phase — when its phosphorylated, that means synthesis phase

Which of the following is true of melting of the DNA (formation of a replicative bubble that separates the two strands of the helix) in eukaryotes? 

It's precise mechanism is unclear

What is the impact of phosphorylation on the ORC?

It releases ORC from the origin and prevents it binding again

The end replication problem refers to difficulties in copying linear chromosomes, this is overcome by the activity of telomerase, which:

Elongates the parental strand at the 3’ end using an RNA template

what do telomeres do?

they act as sacrificial buffers

The need for a 3' hydroxyl group to catalyze DNA synthesis is problematic because RNA primer removal leaves a gap at the end of chromosomes, this occurs on which strand, or strands: 

I. leading strand

II. lagging strand

II

The Hayflick limit states that cells stop dividing after

60 divisions

Overexpression of telomerase can increase the longevity of mice. However, there is concern over replicating this approach in humans, apply your knowledge of telomere biology to indicate why there is concern:

Overexpression of telomerase could lead to lengthening of DNA ends and uncontrolled cell division

what types of cells have a lot of telomerase?

cancer cells and infant cells

what is the function of hTERT and hTERC?

TERT is the protein engine of the enzyme; TERC is the RNA template TERT uses to correctly build DNA repeats

telomere

structure that protects loss of coding information during DNA replication

telomeric repeats

non-coding dna — influences how many times a cell can divide

telomerase reverse transcriptase

protein with RT activity required to elongate template (G) strand

what is pol-a primase?

it is an enzyme complex that synthesizes DNA on C-strand

RNA primer

provides 3’OH group for DNA synthesis on lagging strand

embryonic cells

differentiating cells that multiply to produce a fetus

heart muscle cells

specialized cells that contain lots of mitochondria to prevent fatigue

do RBCs have a nucleus?

no they are anucleate

what should you do if given coding dna strand?

just switch the T to U then use codon chart

what are causes of mutations

errors in DNA replication, damage, imperfect DNA repair

origin of repetitive sequences

copied from a short RNA template during development

how much exposure to chemical is required to guarantee development of cancer?

5 years

first experiment of toxins and cancer

Repeated application of coal tar onto the ears of rabbits

The mutagenic potential of a compound can be quickly screened using the Ames test, from the text, which of the following statements describe the outcome of the test.

In a positive result, bacterial mutants gain the ability to GROW in the ABSENCE of histidine confirming a compound is MUTAGENIC and therefore potentially carcinogenic

what three pieces of evidence are required to define a carcinogen?

short-term cell assays (ex: Ames test —> positive), long term animal tests, epidemiology

potential outcomes of DNA damage

damage gets repaired, mutation is introduced, cell death (apoptosis)

mismatch repair

DNA polymerase (3’ to 5’ exonuclease activity), matching incorrectly inserted bases

nucleotide repair

a vital cellular pathway that fixes bulky, helix-distorting DNA damage caused by ultraviolet (UV) light and chemical carcinogens

base excision repair

remove damaged base and repair nucleotides

DNA glycosylase

does first 3 steps of BER, scans for damaged bases, recognizes specific wrong base (G* for exampe), cuts glycosidic bond between base and phosphate backbone, leaves AP site (empty)

AP endonuclease

removes sugar phosphate backbone

DNA Pol 1

remove primers and add individual nucleotides — BER: synthesize new DNA by adding the correct nucleotide(s) in the gap, using the undamaged complementary strand as a template

DNA ligase

seal the gap; forms new bond at 3’-OH

deamination

spontaneous loss of amino group — cytosine becomes uracil

reactive oxygen species

they can react/attack guanine to make 8-oxoguanine to pair with adenine, transversion mutations opor (high oxidation stress)

dna alkylation

addition of large chemical compounds to the DNA backbone that frequently cause replication fork stalling and double stranded DNA breaks

benzo[a]pyrene

its the one found in grilled/charred meats, leads to mutations, can lead to G —> T mutational signature

UV damage

leads to creation of pyrimidine dimers —> replication stalling forms lesion that blocks replication —> will lead to dsDNA breaks

PCNA

sliding clamp that holds DNA polymerase onto DNA strand itself, helps regulate which DNA polymerase is associated; gets ubiquitinated with DNA stress/damaged + TLS pol becomes attached

RFC (replication factor c)

used ATP to pry the PCNA ring open, place it around the DNA at the primer-template junction, and then close it around the strand

translesion dna polymerase

-able to bypass blockage, involved in DNA replication

-error prone, attaches to PCNA, can throw extra adenines to help with thymine dimers w/ mutations

ubiquitin

acts of a signal to regulate protein-protein interactions (switch out DNA epsilon)

non-homologous end joining

repairs double stranded breaks without a template, fast, can introduce mutations

somatic mutation theory of cancer

process of tumor development do mutations in DNA repair genes occur in the beginning or early stages of formation of a tumor; cancer requires 4 or more genes

how does air pollution cancer?

by stimulating an inflammatory response that activates cells with pre-existing mutations

retinoblastoma

inherited recessively and tumor suppressor gene

cell cycle checkpoint

decision point in cell cycle where favorable conditions must be met to continue —> progression or termination

E2F

activates transcripton of genes involved in DNA replication

CDK (cyclin-dependent kinase)

always present throughout the cell cycle requires cyclin for activity

cdk-cyclin complex (active)

phosphorylates proteins to overcome cell cycle checkpoints

RB gene

inhibits cell cycle progression

G1

time when cell is preparing for DNA synthesis

synthesis phase

DNAis being replicated

what does rb do for cell cycle?

it regulates it and inactivates it

viral-induced tumors

Retroviruses can pick up fragments of proto-oncogenes from host genomes and cause cancer during subsequent infections

mutations leading to formation of oncogenes

gain of function

which repairs are imperfect vs perfect?

nucleotide exicison repair (NER) and homologous recombination are PERFECT; TLS and non-homologous end joining are IMPERFECT