mol bio exam 3

what is a glycosidic bond

bond between sugar and base

how is a dinucleotide formed

sugar’s 3’OH group attacks phosphate group in an atp catalyzed fashion

what is this?

adenonine

what is this?

thymine

what is this

guanine

what is this

cytosine

steps of dna replication (general)

initiation at multiple ori; helicase opens bubble; primase (pol alpha) makes primer; dna poly extends 3’ end; topoisomerase prevents supercoiling; rna primers removed and replaced by dna polymerase; ligase seals gaps

leading strand replication

pcna clamp holds dna pol epsilon synthesizes leading strand

• moves toward helicase

lagging strand synthesis

moves away from helicase incrementally; ssBP bind ssdna to prevent degradation;  pol delta synthesizes lagging strand; ligase ligates nicks in okazaki frags

bacterial replication

signle oriC; dna mediated distortion opens + unwinds DNA; helicase moves 5’ to 3’ on lagging strand (euk go 3’-5’); primase and pol III are recruited; 2nd pol III recruited for lagging strand

bacterial replication termination

only one ter site —> pol III replisomes collide nad dissociate —> topoisomerase and dna gyrase unlink the products

antibiotic target

dna gyrase bc diff enough from topoisomerase

req for antibiotic resistance

mutation changes structure of antibiotic target (making it less fit)

• genetic change must occur

• change must be inherited

• selective pressure req

how do mutations prevent antibiotic use

1. change the target site

2. change uptake into the cell (ex: efflux pumps)

- easy to make changes to genetic code bc of operons + easy uptake of genetic material between bacterial cells

what happens if rna primers remain

lagging strand and maybe leading strand will get degraded

how does proofreading work

1. dNTPs enter DNA pol active site

2. recognizes correct BP via minor groove interaction via H bond formation

3. wrong BP unbinds active —> editing site and is cut by exonuclease

Challenge: tautomers resemble other bases and lead to nonstandard BP

role of M2+ in active site of dna pol

to allow negative charges of phosphate groups to come tg

topoisomerase 1

cuts ssDNAt

topoisomerase 2

cuts dsDNA

how are histone mod patterns preserved

1. g1/s phase —> synthesize histones

2. make histone modifications before fork is made (dna pol wont be affected and wave of euchr before fork)

3. h3/h4 tetramers stau associated while h2a/h2b unbind and rebind (w random strand)

4. chromatin remodeling complexes aid w histone displacement

how does telomerase work

1. binds 3’ end

2. adds dNTPs w bound RNA template (repeats many times for long extension)

3. primer is added near 3’ end 

4. dna poly and ligase

homologous recomb steps

1. spo11 cuts

2. strand invasion via rad11

3. additional dna synth

4. cleavage

BER summary

1. glycosylase flips out base and cuts glycosidic bonds

2. AP endonuc and phosphodiesterase remove sugar phosphate

3. dna poly and ligase (may replace a flap)

NER steps

1. excision nuc cuts strands

2. helicase separates them

3. cut piece is released

4. poly and ligase

transcription coupled NER

rna pol II pauses at damage

backs up

repair mechanism

MMR

1. MSH binds to mismatch site

2. MLH cuts backbone of newly synth (nascent strand)

3. strand removal

4. dna pol + ligase

HDR steps

1. dsDNA break —> overhang creation

2. rad51 facilitates invasion

3. dna pol extends using template

4. uninvasion + ligation

5. MMR as needed

NHEJ

1. Ku heterodimers bind broken ends

2. nt removed during processing —> overhangs

   1. join endsin

innate immunity

aggresssive and broadly reactive

immediate detection of intruders

adaptive immunity

intelligent and specific

tailored response

aggressive or suppressive

rag complex

made of rag1 + rag2 binding to rss motifs

• catalyze ds breaks between rss and gene segment

• proper alignment may req looping

mechanism of vdj recombination

1. rag complex binds to rss motifs + catalyzes dsDNA breaks

2. creates hairpins at coding ends

3. endonuc, artemis are recruited and activated by dna pKcs —> cleaves hairpin loop and generates overhangs

4. Tdt randomly adds non templated nucleotides

5. NHEJ using Ku proteins and ligase —> RSS ends turn into signal joint

what are piRNAs

silence transposable elements and help maintain genomic stability 

longer than mirRNAs

bound by piwi proteins 

transcribed by uni/ bidirectional transposons

function of pirnas

allow transposon rna degradation (ex: post-transcriptional silencing)

can direct dna methylation of transposon/ repetitive elements

relevance

important in germline + stem cells

affect fertility

implicated in cancer

pirna vs mirna

mirna req dicer

pirna can act pre/ post transcriptionally

transposition

transposase —> loop + ds break to remove transposon in donor chromosome

• ds break repaired via nhej

new loc is cut and transposon added