MM exam 1

anabolic

energy → complex molecules

catabolic

complex molecules broken down → energy

phosphodiester and glycosidic

sugar -phosphate

sugar to nitrogen base

Purine de novo pathway

assembled on ribose

PRPP

PPAT, glutamine, THF

IMP intermediate

NTP/ dNTP

Pyrimidine de novo pathway

PRPP

Carbamoyl phosphate/ aspartate

Orotate ring + PRPP via ORPT

OMP intermediate

NTP/dNTP

THF, glutamine, PPAT function

methyl donor

nitrogen donor

1st committed step

feedback loops to prevent waste/ nucleotide imbalance

uracil → cytosine

Pyrimidine salvage pathway (thymine)

Thymine

thymidine phosphorylase + deoxyribose

thymidine

kinase

dTMP

how can RNR be regulated

positive feedback loops

• no energy/ RNR no dNTP

negative feedback loops

• excess U → T ; no dividing

thymidylate synthase

dUMP

thymidylate synthase enzyme

THF

dTMP

AZT

Thymidine analog interacts with thymine kinase can stop HIV replication via reverse transcriptase/ chain termination

what can inhibit thymidylate synthase

methanotrexate as a competitive inhibitor mimics DHF

5FU engaging with THF as a suicide inhibitor

Excess uric acid comes from

Treatment

HGPRT deficiency

PPAT deregulation

High purine diet

Suicide inhibitor on xanthine

Intiation for Prokaryotes

DnaA bind to oriC

HU melts helix, bubble

DnaB helicase unwind via ATP hydrolysis

ssbinding proteins

DnaG primase form primosome

Initiation for Eukaryotes

ARS binds B3, B2 region melts

MCM complex → DNA

CMG helicase for unwinding via ATP hydrolysis

RPA as ssB proteins

ARS eukaryotes complex

A and B1 ORC

B2 DNA helix unwinding

B3 binding for ARS

clamp loader

beta clamp

bring pol to clamp

tether polymerase to DNA

DNA pol 3 core

alpha = polymerase

epsilon = 3’ → 5’ exonuclease

theta = structural component, simulates epsilon

other greek subunits mold clamps on

clamp loader complex prokaryotes

tau links pol 3/ DnaB

chi interact with ssB to release primase

Psi bridge between tau/ gamma

gamma open/close beta clamp via ATP hydrolysis

delta opens beta clamp

delta prime keeps delta in place

steps of a beta clamp

ATP → gamma complex on clamp loader

engage with beta clamp to open

closes around DNA primers

ATP hydrolysis closes beta clamp around DNA

DNA pol 3 replaces clamp loader

Elongation for prokaryotes

topoisomerase

PCNA tether pol 3

RFC loads PCNA to template

DNA pol 3 holoenzyme

DNA pol 1 exonuclease

elongation for eukaryotes

topoisomerase

DNA pol ϵ (leading) and DNA pol δ (lagging) with proofreading

PCNA tethers

RFC clamp loader

Flap endonuclease 1 remove primers/ Okazaki fragments

DNA ligase

RFC subunit eukaryotes

RFC1 = bind to open PCNA ring

2-4 = loads PCNA to primed DNA

5 = closes ring

ssbinding proteins prevent what

hairpins, nuclease attack, reanealling

termination for prokaryotes

terminator sequences opposite w/ Tus proteins to trap replication fork

no more ssB proteins

concatenated daughter circles

type 2 topoisomerase IV unlinks

termination for eukaryotes

2 replication forks collide

CMG removed

5’ end shortening problem

Telomerase w/ RNA template extend overhangs make T-loop

shelterin function

protect telomeres from NHEJ and overhang loss

what can go wrong

mismatched bases → deformed helix

replication slippage on lagging strand → neurodegenerative diseases

UV damage → thymine dimers damage backbone/ stall machinery

ds breaks from radiation → chromosomal loss

telomere loss after primer removal → chromosomal end fusions

how does DNA repair alliviate these problems

exonuclease

post replication enzymes

nucelotide excision on bulky dimers

kinase reads ds breaks, pause cell cycle/ allow repair/ apoptosis

telomere extension replaces lost sequences prevent non homologous end joining

Lac/ Trp

lac binding to operator in absence

Low glucose, cAMP and CAP binding near promoter for transcription

Trp bind when in excess

CAP

Acts as a central energy sensor. When glucose levels drop, intracellular cyclic AMP (cAMP) rises and binds to CAP. The active cAMP-CAP complex binds upstream of promoters (like the lac operon) to physically interact with RNA polymerase and drive high-level transcription

eukaryotic regulatory elements

enhancers influence transcription factors to recruit DNA pol 2 position

insulators block enhancer from activating a non-target promoter

repression proteins bind to operators to prevent transcription

Consensus

DNA pattern just before a gene that acts platform for proteins to turn gene expression on or off (TATA box)

DNA affinity chromatography

Purifies/ isolate transcription factors for mass spectrometry or characterization

DNA binds to motif on beads down a column

mixture passes through to rid non-binding protein

salt eluts target DNA

electrophoresis mobility shift assay

checks if a protein interacts with a DNA sequence

Evaluate binding specificity/ affinity using competitor DNA or sequence mutations

bind tag to DNA

control just tag

DNA + tag prove binding

DNA + tag + antibody confirms protein interaction

chromatin immunoprecipitation (ChIP)

using protein of interest inside living cells and pull out genomic DNA it was bound to

lock DNA + protein together with formaldehyde

lyse and break genome via sonication

antibody binds to protein to take out DNA, wash debris

heat/ enzyme away the protein from DNA

use downstream detection to map DNA sequence/ all binding sites on genome

western blotting

using antibody to measure protein abundance in the cell

lyse/ denature proteins via SDS
electrophoresis

transfer to membrane w antibodies to bind

soak in BSA to prevent non-specific protein binding

use chemiluminescence to find protein abundance

TATA box significance

allows housekeeping genes to be transcribed easily via TATA binding protein/ sigma factors