Principles of Molecular Biology 1

What are the (4) criteria for genetic material?

must replicate, store information, express information, and allow for variation through mutation

What do nucleotides consist of?

a nitrogenous base, pentose sugar, and phosphate group

Pentose Sugars

ribose (with OH group) and deoxyribose (without OH group)

Pyrimidines

six member rings (one ring)

includes cytosine, thymine, and uracil

Purines

nine member rings (two rings)

includes adenine and guanine

Nucleotides vs Nucleosides

nucleosides - no phosphate group

nucleotides - have a phosphate group (more energy)

Functions of Nucleotides

form DNA/RNA, energy carriers, cell signaling, and enzyme cofactors

Pentose Sugar Carbons

1’ attaches to the nitrogenous base

2’ contains -OH in ribose and -H in deoxyribose

3’ and 5’ - connection points between consecutive nucleotides by a phosphate group (provide direction)

Chargaff’s Rules

base composition using chromatography

amount of A=T and amount of C=G

sum of pyrimidines equals the sum of purines

percent of C+G does not equal the percent of A+T

DNA model

A double helix

Two antiparallel strands connected by base pairing

Stacked nitrogenous bases

Nitrogenous bases of opposite chains are paired

Major groove alternates with minor groove

10 bp per rotation

B DNA is the most stable of DNAs under physiological conditions

Why Uracil in RNA

allows for modifications - three types of RNA (rRNA, tRNA, mRNA)

2’ OH of ribose

forms H-bonds better than DNA - allows for more interactions between proteins and nucleotides, unique structure (A type helix), more modifications, formation of ribozymes

Features of bacterial chromosomes

usually 1 circular chromosome

stored in the nucleoid

bound by positive proteins - nucleoid-associated proteins (NAP) and histone like proteins (HLP) which bend DNA induce supercoiling

nucleosome

eukaryotic DNA wrapped around an octamer of histones

146 bp DNA

wrapped left-handed

N-terminal tails of histones extend out

core histones

H2A, H2B, H3, H4

2 of each type of histone in each nucleosome

rich in Lysine and Arginine

(are also histone variants which can be exchanged for a core histone and change chromatin structure)

Heterochromatin

highly condensed

few to no genes

replicate late

inactive X chromosomes and much of the Y chromosome

highly conserved and repetitive sequences

can be facultative (variable) or constitutive (permanent)

Euchromatin

“open”

transcriptionally active

modified with histones which recruit recombination proteins

Centromere

middle of sister chromatids (connecting them)

essential for chromosome movement

repetitive sequences

differ in size/sequence between species

histone variants allow binding proteins to form the kinetochore (attaches spindles to pull apart chromsomes)

Telomeres

heterochromatic ends of chromosomes

necessary to prevent DNA degradation

conserved across vertebrates

G and C-rich

have T-loops which protect the ends of chromosomes from degradation

Absorption of UV light

Nucleic acids absorb UV, which is used in the localization, isolation, and characterization of nucleic acids

Molecular Hybridization

denatured (by heat/stress) DNA can become single-stranded, the strands can hybridize to form duplex structures even if they are not from the same source (depending on sequence)

FISH

fluorescent in-situ hybridization

mitotic cells are attached to slides and ssDNA is added, fluorescent probes are used to monitor hybridization

the probes are nucleic acids that hybridize with only certain chromosome areas

Electrophoresis

separates DNA or RNA by size

the strands are sent through aragose gel, where the larger strands experience more resistance and do not travel as far

Semiconservative replication

each replicated DNA consists of one “one” and one new strand

how does DNA replicate

the specific pairing of the bases allows DNA to serve as templates; complementary strands are made using an existing strand as a template

Meselson and Stahl

performed heavy and light nitrogen experiments with replicating E. coli to show that DNA replicates semi-conservatively

What is needed for replication?

a single-stranded DNA template

raw materials (dNTPs: dATP, dGTP, dTTP, dCTP)

DNA polymerases and other proteins

Key issues of replication

unwinding the helix

reducing increased coiling

primer synthesis for initiation

discontinuous synthesis of the second strand

removal of RNA primers

joining the gaps in discontinuous strand

proofreading

4 steps of replication

initiation, (unwinding), elongation, termination

Initiation in prokaryotes

begins at a single origin (oriC)

at the initiation site, DNA is unwound and two replication forks begin in each direction

Replicon

the length of DNA that is replicated

Initiation site in eukaryotes

multiple origins (larger genomes)

Initiator proteins (proks)

DnaA - binds the origin, “loosens” DNA

DnaB - helicase

DnaC - “loader” that attaches DnaB to DnaA

Initiation process (euks)

Origin recognition complex (ORC)

ORC recruits Cdc6 + Cdt1

Loads two ring-shaped helicases (MCM2-7) to DNA

ORC dissociates and accessory proteins interact with MCM2-7

the helicase complex is phosphorylated to start replication

Full helicase complex (CMG)

made up of cdc45, MCM, & GINS

unwinds DNA at the replication forks

a single strand fits in the complex, displacing the other

helicase comparison

Both - AAA+ proteins, 6 subunits

Proks - helicase on lagging strand (5’ to 3’)

Euks - helicase form on leading strand (3’ to 5’)

Single-stranded binding proteins

SSB in bacteria

Replicon protein A (RPA) in euks

bind to the displaced strand of DNA to stabilize it

Priming

DNA polymerase needs a 3’-OH to synthesize new DNA

Primase - provides 3’-OH

Prokaryotes - RNA primer DnaG (10-30 nt)

Eukaryotes - RNA +DNA primer polymerase alpha-primase complex (10 nt RNA and 20-30 nt DNA)

2 things needed to bring polymerase to the DNA template

1. Sliding clamp - anchors DNA pol to the template

2. Clamp loader - set of proteins that opens the ring and loads at the template-primer junction

Prokaryote priming

Clamp established at the 3’ end of the primer

DNA pol III is recruited

Eukaryote priming

Clamp established

DNA pol alpha-primase complex (polymerase switching)

• DNA pol epsilon - leading strand

• DNA pol delta - lagging strand

Elongation

After DNA pol is associated with the DNA

Parental DNA is read by DNA pol

Complementary bases are added to the growing strand in a 5’ to 3’ direction

DNa polymerase hand structure

• fingers - position the incoming nucleotide

• thumb - holds the elongating dsDNA

• palm - active site where the nucleotide is added

Polymerases

many different variations with different functions

grouped according to evolutionary lineage

different organisms have different ones, indicating early evolutionary divergence

Termination

Completion of replication

1. resolving torsional stress

2. meeting of replication forks

3. dissociation of the replisome

Replisome

helicases, primase, DNA polymerase, sliding clamp, and clamp loader

Topoisomerases

add/remove twists in the alpha helix

during replication, they remove twists (fixing torsional stress from elongation)

Prok termination

Tus (termination utilization substance) binds to ter (termination) sites

only move in one direction

Euk termination

replication bubble meet

Okazaki fragment maturation (proks)

DNA pol III dissociates from the strand when it reaches an RNA primer, but the sliding clamp remains associated

DNA pol I removes the RNA primer and replaces it with DNA

DNA ligase seals the “nicks” between the fragments

Okazaki fragment maturation (euks)

DNA epsilon or delta continue to make DNA after they meet the RNA primer

the DNA displaces the RNA primer & DNA from the template, making a flap

Flap endonuclease (Fen1) cleaves the flap

If the flap gets too long, Dna2 cleaves it

DNA ligase seals the “nick”

Replication fork coupling

DNA pol moves in different directions on the lagging and leading strands

The lagging strand loops around so that DNA pol can move with the replication (regulated together)

End-replication lagging strand

problems with replicating the ends of lagging strands

most eukaryotes use telomerase to fix this (but there are other mechanisms)

Telomerase

Synthesizes telomeres - conserved G-rich (3’) and C-rich (5’) ends

Telomerase RNA component (TERC) - guide and template

Telomerase reverse transcriptase - turns RNA template to DNA

Replication error rate

incredibly low

1 in 10 billion bp

Quality control steps (3)

Polymerases have low error rates

Polymerase proofreads the new strands

Mismatch repair (purines vs pyrimidines)

Chromatin remodeling

Chromatin - DNA + histones

Histones are found in particular locations and with varying modifications (methylation)

After replication, nucleosomes repackage

Half H3 & H4 histones go to the daughter DNA and half stay with the parent which allows both strands/cells to inherit the modifications

New histones are modified in line with the old ones

Mutation

any base-pair change, single base-pair substitutions, deletions or insertions of base pairs, or major chromosomal restructuring

can be in somatic or germ cells

can be in coding or non-coding regions

Transition

point mutation

swap a purine for purine OR pyrimidine for pyrimidine

Transversion

point mutation

swap a purine for pyrimidine OR pyrimidine for purine

Missense mutation

results in a new triplet code for a different amino acid

Nonsense mutation

results in a new triplet code for a stop codon (stopping translation prematurely)

Silent mutation

results in a new triplet code for the same amino acid

Frameshift mutations

insertions or deletions of base pairs

cause a shift in the reading frame

triplets’ reading frames are changed during translation

Loss vs gain of function

loss of function - reduces or eliminates product activity

gain of function - increases, adds, or misregulates activity

Somatic vs germ mutations

somatic cells - create cellular mosaics and can cause cancers

germ cells - can enter gametes and pass down to offspring

Endogenous sources

Internal causes for mutation - replication error, oxidation, or hydrolysis

Called spontaneous mutations

Exogenous sources

External causes for mutations - radiation and chemical mutagens

Called induced mutations

Spontaneous mutations

Naturally occurring changes in nucleotides

Arise from biological processes or chemical processes that alter bases

Very low mutation rates, but vary between species and genes

Mutagens

any agent that induces mutation

fungal toxins, cosmic rays, UV light, industrial pollutants, X-rays, chemicals from tobacco smoke, or IR

Tautomeric shifts

Tautomers of the nucleotides - some of the H atoms change position

Creates isomers

can form atypical base pairs, so incorrect nucleotides can be incorporated easily

Ribonucleotides

DNA pol frequently incorporates ribonucleotides in place of deoxyribonucleotides

More frequent in euks

Use the Ribonucleotide Excision Repair (RER) pathway to fix these errors - RNase H2

(If RER is not working strand breaks, mutagenesis, and neurodevelopmental disease can occur)

UV mutations

UV light promotes the formation of pyrimidine dimers

Perturbs stacking between DNA bases, distorts the phosphodiester backbone, and prevents replication

Depurination

loss of a purine, resulting in an apurinic site

(can also lose pyrimidines, but less common)

Deamination

loss of the amino group from a base (NH2)

DNA alkylation

addition of alkyl groups to DNA bases

ROS

reactive oxygen species

produced by normal cell metabolism, but can alter bases, sugars, and the backbone

oxidized guanine produces transversions

antioxidants and DNA repair are able to limit the damage burden

Mismatch repair system

uses the other strand as a template to fix replication errors

mismatches are recognized by the MutS protein

the mismatch on the new strand is fixed, not the old strand (recognized because new DNA is not methylated)

can also fix insertions and deletions (slipped strand misreplication)

Stalled replication forks

DNA pol is stalled when a lesion (damage/error) is encountered

Translesion polymerases trade fidelity for the ability to copy damaged DNA (TLS bypass)

Template switching uses the sister duplex to avoid copying the lesion (TS avoidance)

Direct reversal

photolyases reverse pyrimidine dimerization (not placental mammals)

base and backbone methylation - methyltransferase

alkyltransferase

uses hydrolases to covert damaged nucleotide precursors into dNMT (methylated)

Base excision repair

replaces a single nucleotide (or occasionally a couple)

glycosylase recognizes the base and removes it

AP endonuclease cleaves the phosphodiester bond

DNA pol replaces the base and seals the nick

Largely conserved in proks and euks

Nucleotide excision repair

replaces largers lesions (10-30 bp)

In bacteria:

Uvr A & UvrB recognize damage

UvrB unwinds the damaged region

UvrC nicks the damaged DNA

UvrD removes DNA

DNA pol and ligase replace and fix area

DS break repair

Homologous recombination repairs during late S or early G2 phase

Nonhomologous end-joining is activated in G1 (can be any time in the cell cycle)

SOS response

widespread DNA damage in bacteria

RecA - key SOS protein

LexA - inhibits cell cycle to give time for repair

Eukaryote DNA damage sensors

Replication protein A (RPA) - ss breaks

MRN & KU - ds breaks

ssDNA repair (euks)

RPA senses ssDNA

usually removed during replication, but accumulates if the fork stalls (indicating damage)

recruits ATR (reg kinase) via ATRIP

also recruits a specific sliding clamp (9-1-1)

9-1-1 recruits TOPBP1 to activate ATR

dsDNA breaks

recruitment of MRN can lead to NHEJ or HR

in HR, a homologous duplex is needed to be used as a template

if repairs cannot be made, p53 mediates cell death