biochem exam 4

DNA is an antiparallel ____ of _____

DNA is an antiparallel ____ of _____

dimer

Dna nucleic acid strands

deoxyribose is missing the ____ at the _____ position

hydroxyl group

2'

how are the chains of DNA polymerized

phosphodiester linkage from 3'-hydroxyl of one ribose to 5'-hydroxyl of next ribose

DNA is a _________ of _________ with _______ and ______ bases attached to _____ of the _______

duplex linear polymer

deoxyribose 3',5'-phosphate

purine

pyrimidine

carbon-1'

deoxyribose subunit

how do the bases form the DNA "rungs"

hydrogen bonding

direction of DNA synthesis

5'-end to 3'-end

denaturing

heat induces reversible dissociation of base pairing that unwinds double strand --> single strand

sigmoidal curve

reannealing

complementary nucleotides reanneal to reform their original base pairs

nucleosome

DNA coils around a histone octamer

histones --> chromatin --> chromosome

chromatin

DNA-RNA protein complex

chromosome

compact, highly organized, 23 pairs

linker histone proteins

H1/H5

core histone proteins

H2A, H2B, H3, H4

how many DNA bases are wound around each histone core

140-150

how many bases do linker DNA have

40-60

histones

highly alkaline proteins found in eukaryotic cell nuclei that package and order DNA into nucleosomes

chief protein components of chromatin

act as spools around which DNA winds

play a role in gene regulation

human chromosal DNA

3.3 x 10 bp

23 DNA molecule in haploid, 46 in diploid

20,000 - 30,000 genes encoded

density 1 - 40,000 bp

introns frequently found in most genes

~3% coding DNA

universal genetic code

mendelian inheritance for autosomes and X chromosomes

mitochondrial DNA

16,569 bp

several thousand DNA molecules per cell (polyploidy)

37 genes encoded

density 1 - 450 bp

introns absent

~93% coding DNA

AUA and TGA, AGA and AGG stop codons

exclusively maternal inheritance

AUA

methionine

TGA

tryptophan

AGA, AGG

stop codons

cell cycle phases

Interphase (G1, S, G2)

Mitosis (M)

G1 phase

prior to DNA synthesis

RNA and protein synthesis occurs

S phase

DNA replication occurs

G2 phase

post DNA synthesis

DNA repair

mitosis preparation

M phase

mitosis

cell divides into two daughter cells

why does DNA replication occur in the S phase

so the genetic material of the cell can be doubled before it enters mitosis or meiosis, allowing there to be enough DNA to be split into daughter cells

what triggers DNA replication or synthesis

helicase binds to double-stranded DNA and pries the two strands apart, breaking the hydrogen bonds between the bases

helicase

break weak hydrogen bonds between bases to template (single strands with unpaired bases)

SSB

keep single strands apart to prevent reannealing

primase

produce RNA primer to initiate DNA synthesis

makes Okazaki fragments

DNA polymerase III

binds leading strand of DNA nucleotides starting at 3' end of RNA primer to 5' end

synthesizes new DNA strand from 5' --> 3'

proofreading capability by excising incorrect nucleotide and replacing with correct ones

DNA polymerase I

takes off RNA primer so that DNA polymerase can replace it with DNA nucleotides

ligase

inserts phosphate into any gaps in the sugar-phosphate backbone of the DNA strand --> removes DNA nick (gap)

Okazaki fragments

between 1000-2000 nucleotides long in prokaryotes

100-200 nucleotides long in eukaryotes

separated by 12-nucleotide RNA primers, unligated until RNA primers removed

ligase connects Okazaki fragment onto (now continuous) newly synthesized complementary strand

why not make a full strand of DNA instead of fragments?

while waiting for DNA to unzip a whole strand, self binding will prevent further replication

is RNA priming used on both strands?

yes, only one RNA primer needed for DNA polymerization on leading strand

several RNA primers needed for lagging strand because DNA polymerization direction is opposite to replication fork displacement

why is RNA primer necessary?

DNA polymerase requires free 3'OH so first RNA primase is required to generate RNA primer

then DNA polymerase uses 3'OH end of annealed RNA primer to start copying complementary strand

why are DNA primers not used?

there are abundant ribonucleotides in the cell compared to deoxyribonucleotides and evolution favors mechanisms that are energetically less expensive

why are there multiple origins of replication in eukaryotes?

eukaryotic chromosomes are larger (contain 60x more DNA than prokaryotes) so multiple origins are needed to replicate the entire chromosome in a short amount of time

DNA replication rate in humans

40-50 nt/sec

1-2 months for a single round of replication for the whole genome

replication origin

site where replication begins

replication fork

site of DNA unwinding, where helicase binds to

replication bubble

separations of DNA strands

polymerase chain reaction (PCR)

used to detect or amplify certain DNA fragments on target DNA template

three types of reverse transcription

retrovirus infection and reverse transcriptase (RT)

anti-retroviral chemotherapeutic agents (targeting RT)

RT-PCR

reverse transcriptase

RNA-directed DNA polymerase, enzyme encoded from genetic material of retroviruses

catalyzes transcription of retrovirus RNA --> complementary DNA (cDNA)

fundamental component of RT-PCR

transcription

use DNA as template to synthesize RNA

DNA is copied into mRNA by RNA polymerase

reverse transcription

use RNA as template to synthesize DNA

RNA is copied into DNA by reverse transcriptase

viral genome replication

use RNA as template to synthesize DNA

how does anti-viral therapy work?

targets reverse transcription

retrovirus

single strand RNA genome contained within a protein shell that enclosed in a lipid envelope

three genes that make up a retrovirus genome

group-specific antigen gene (gag)

polymerase gene (pol)

envelope gene (env)

three enzymes that pol gene encodes and what it does

protease, reverse transcriptase, integrase

catalyzes the steps of retroviral infection

retrovirus infection process

protease mediates retrovirus entry into host cell

reverse transcriptase catalyzes retroviral RNA --> proviral DNA (retrovirus hijacking host's genetic transcription machinery to construct DNA provirus)

integrase initiates insertion of proviral DNA into host DNA

two types of enzyme activity of reverse transcriptase

polymerase activity

nuclease activity

polymerase activity

RNA-directed DNA polymerase, lack of proofreading ability

nuclease activity

removes RNA, single strand DNA is used as template to synthesize double strand complemental DNA

what is central to the infectious nature of retroviruses and what kind of diseases do they cause

reverse transcriptase

HIV (AIDS), HTLV-1 (leukemia)

2 chemotherapeutic agents targeting RT

nucleoside RT inhibitors (NRTIs)

non-nucleoside RT inhibitors (NNRTIs)

nucleoside RT inhibitors (NRTIs)

deoxynucleoside analog (mimic substrate), requires activation by phosphorylation

incorporate themselves into virus DNA --> compete against natural nucleotides --> stop reverse transcription process --> resulting DNA is incomplete and cannot create new virus --> blocks HIV replication and infection of new cells --> no effect on already affected cells

first treatments available to people living with HIV

drugs that inhibit reverse transcriptase

AZT (zidovudine) = first drug approved by FDA to proloterm-63ng lives of AIDS patients, terminates proviral DNA chain before the enzyme can finish transcription

non-nucleoside RT inhibitors (NNRTIs)

non-competitively bind to reverse transcriptase itself and alter its shape, blocking its function

bind to enzyme at different site from NRTIs

do not require activation by phosphorylation

stops HIV production by preventing conversion of RNA --> DNA

often given in combination with NRTIs

RT-PCR (description and process)

powerful tool used in research and in diagnosis of diseases (cancer) by detecting gene copies (mRNA level) in the target cells/tissues

1. total RNA isolation (remove DNA)

2. reverse transcription: RTs use RNA template and short primer complementary to 3' end of RNA to direct synthesis of first strand of cDNA, RT Rnase activity removes RNA from RNA-DNA complex and RTs synthesize complementary cDNA

3. regular PCR with cDNA

allows detection of low abundance RNAs in sample and production of corresponding cDNA which facilitates cloning of low copy genes

stimuli for DNA damage and repair

ionizing radiation (UV or radioactive)

reactive oxygen species (ROS)

alkylating agents (carcinogens)

targets for DNA damage and repair

base, sugar

4 ways to repair DNA damage for nucleotides

modification (oxidation, dimerization)

deletion

sequence inverseion

transposition

mutations

changes in the genetic message

base excision repair (BER)

recognizes damaged bases that do not cause a signification distortion to DNA helix

corrects DNA damage from oxidation, deamination, and alkylation

DNA glycosylase removes damaged base, AP endonuclease cleaves DNA near site of defect, DNA polymerase fills gaps, DNA ligase seals the nicks

DNA glycosylase

removes damaged base in BER

AP endonuclease

apurinic/apyrimidinic endonuclease cleaves DNA near site of defect in BER

DNA polymerase

fills the gap in BER

DNA ligase

seals the nicks in BER

which polymerases do eukaryotes and prokaryotes use in BER

eukaryotes: DNA polymerase beta

prokaryotes: DNA polymerase I

what are the downstream of BER utilized to repair

single-strand breaks

nucleotide excision repair (NER)

recognizes damaged regions based on their abnormal structure as well as on their abnormal chemistry

excises and replaces them

1. DNA damage occurs

2. recognitions of damaged DNA (nucleotide)

3. DNA double strands are separated and SSB keeps them apart

4. endonuclease (ERCC1-XPF) removes damaged DNA

5. gaps filled by DNA polymerase

6. nicks sealed by DNA ligase

mismatch repair (MMR)

recognizes misincorporated nucleotides, repair errors that occur during DNA synthesis

plays key role in maintaining genomic stability

does not operate on bulky adducts or major distortions to DNA helix

most mismatches are substitutes within a chemical class (C instead of T), causing only subtle helical distortions in DNA and misincorporated nucleotide is a normal component of DNA

MutS

enzyme that distinguishes normal base pairs from those resulting from misincorporation in MMR

how does UV induce DNA damage

forms thymine dimer between adjacent nucleotides

UV-induced DNA repair through NER

1. formation of DNA dimer

2. recognition of dimer and DNA cut

3. excision of dimer

4. gap filled by DNA polymerase

5. nick sealed by DNA ligase

photolyase

DNA repair enzyme that breaks dimers during UV-induced DNA NER repair

requires visible light (>300nm) to function

2 changes caused by oxidative damage of guanosine, risk factors

damage to base

mismatch

smoking, aging, atherosclerosis, diabetes

two ways to introduce 8-oxoG into DNA

1. base in free nucleotide is oxidized (MTH1 in cytoplasm and mitochondria), then incorporated into DNA during replication (G2)

2. base is oxidized in the nucleotide in DNA and repaired through BER (OGG1) and MMR (MYH), results in A/T transversion mutation if not repaired before second round of DNA replication (S)

single strand break (SSB)

poly ADP-ribose polymerase 1 (PARP-1) mediated repair via BER machinery

double strand break (DSB) repair pathways

homologous recombination (HR) - error-free repair, S/G2 phase

non-homologous end joining (NHEJ) - error-prone repair, G0/G1 phase

what do cells need to repair DNA and survive (2)

BER or HR machinery

will die without

NHEJ

results in gene mutation

most significant of external agents that induce DSBs

ionizing radiation

which mutations predispose for development of breast and ovarian cancer, and what are the percentages

germline BRCA1/BRCA2

breast cancer 87%

ovarian cancer 50%

synthetic lethality interaction

between HR deficiency and PARP inhibitors, new therapeutic strategy to treat cancer cells with HR deficiency

HR deficiency predisposes to cancer development but sensitizes cancer cells to DNA damage-inducing therapy

general classes of RNA, percentages, and functions

rRNA (80%, form ribosomes)

tRNA (15%, adapter)

mRNA (5%, directs synthesis of cellular proteins, aka gene copy)

structure of RNA

single strand + hairpin loop

how many subunits do eukaryotic ribosomes have

two: large (60S) and small (40S)

anticodon loop

complimentary codon to mRNA

acceptor stem

3 unpaired bases for all tRNA at 3'-end (CCA)

tRNA

adaptor/translator between each codon and its coded amino acid, carries/charged by amino acid to ribosome for protein synthesis

AA converted to aminoacyl form, added to 3'-acceptor stem of uncharged tRNA

prokaryotic mRNA

mRNA transcribed in cytosol/cytoplasma

polycistronic genes

no introns

no splicing

naked mRNA (no modifications at 5' and 3' ends)

eukaryotic mRNA

mRNA transcribed in nuclei and exported to cytosol (necessitates RNA processing for stability)

not polycistronic

contains exon and intron (intron spliced out during RNA splicing)

modified 5' and 3' ends (5' capping and 3' polyA tail)

DNA polymerase (template, substrate, product, require primer, proofreading)

DNA

dNTP

DNA

yes

yes

RNA polymerase (template, substrate, product, require primer, proofreading)

DNA

NTP

RNA

no

no