Genetics Mod 2,DNA Structure and Replication

DNA Structure and Replication

historically scientists knew these had to be the characteristics of genetic material

information (contain necessary info to make entire organism & specify traits, replication/transmission (it must be copied and passed from parent to progeny), and variation (must be capable of changes)

if no functional alleles in organism how will transcription be carried out?

no transcription or if transcription occurs it will not produce a functional gene product ∴ no functional transport protein

list of finite molecules that were POSSIBILITIES to be basis of gene material

proteins, lipids, nucleic acids (DNA & RNA), carbohydrates

idea behind Griffith’s 1928 bacteria experiment to develop vaccines

take a sample of the infectious agent and subject it to fatal conditions for the bacteria (heat by boiling) and inject it into animal & animal develops a resistance w/o chance of infection

Avery, McLeod, & McCarty expanded on griffith’s experiment by

testing all of the potentials for basis of gene material and determining that DNA was the transforming agent because it was the only component not destroyed and did not recover the infectious ability of the bacteria

Hershey Chase Experiment (1952): uses a specific type of virus:

bacteriophage that lands on surface (majority of virus will remain on outside of bacterium), injects genetic material into interior, genetic material is copied within bacterium and creates pieces of virus that comes together to infect bacteria

hershey chase experiment cont. with radioactive markers process

radioactively marked phage particle introduced to E.Coli to dock and inject w/ genetic material to interior, and the husk/remainder separated from surface of bacteria through sheer force (blender)

when radioactive isotope of sulfur is used in bacteriophage, where is sulfur found

w/ the phage ghosts

when radioactive isotope of phosphorus is used in bacteriophage, where is phosphorus found

isolated to the injected bacterial cells

how were the phages marked in the hershey chase experiment

phages grown in media containing radioactive isotope and allowed to reproduce in bacteria, phages are isolated, phages grown in a way so majority if not all atoms (sulfur or phosphorous) w/in phage contain radioactive isotope

what implies that nucleic acids are the basis of genetic transfer of material

molecules containing Phosphorus (component of DNA) were injected with the genetic material from the bacteriophage virus

what implies that proteins contain sulfur

molecules containing sulfur were not injected with the genetic material from bacteriophage

combinaion of what two experiments led to the scientific community adopting the theory that DNA was basis of genetic material

hershey-chase & griffiths

what organisms use RNA as genetic material

no known examples of cell-based life forms doing so but some viruses do

nucleic acids are…

large, class of macromolecule (like RNA & DNA) overall structure is a polymer, nucleotides make up these molecules

polymer

long, linear molecule that has individual subunits that are chemically similar to each other (ie monomers ie nucleotides)

when wrapped around protein structures what does a double helix form

a condensed three-dimensional structure

three components of a nucleotide

phosphate group (can be multiple), pentose/ribose sugar (5-carbon sugar), and a nitrogenous base (ACTGU)

nucleoside

nitrogenous base + ribose (sugar)

nucleotide

nitrogenous base +ribose (sugar)+ phosphate group (1 or more)

meaning of NTP or dNTP

“any” nucleotide triphosphate

where does the phosphate group attach to on a nucleotide

5’ carbon

where does the phosphate group attach to on a polymer/polynucleotide

3’ OH (hydroxyl)

what differs between deoxyribose and ribose sugar

2’ position, deoxy missing OH (hydroxyl) ribose has OH

purines and ring system

adenosine (A) and guanine (G), bicyclic = two rings

nitrogenous bases are always attached to ribose/deoxyribose at what position

1’ carbon position

pyrimidines and ring system

cytisine (C) and thymine (T), monocyclic = one carbon ring

what type of bonds keep nucleotides attached to each other in a single strand

covalent bonds

what type of bonds keep the two strands attached to each other in a double helix

hydrogen bonds

where are nucleotides connected to form a polymer of DNA and what process occurs during this connection

dehydration synthesis allows the 5’ end of one nucleotide to join to the 3’ end of the next nucleotide to form a polymer through a phosphodiester linkage

what are nucleotides called after they make a phosphodiester linkage to another nucleotide

nucleotide residue

phosphodiester linkage

connection of two nucleotides from phosphate group

base pair of guanine (G) and how many hydrogen bonds

cytosine (C) and 3 THREE hydrogen bonds

base pair of adenine and how many hydrogen bonds (DNA)

thymine and 2 TWO hydrogen bonds

what are two bases called once they bind

complements (of each other)

DNA polymerase III catalyzes…and requires

DNA synthesis and requires a DNA template all four dNTPs (any of the ACTG nucleotides)

key points that show limitations of all known DNA Polymerases

incoming nucleotides are selected because they are complementary to the template, incoming nucleotides are only added to the 3’ end of DNA, nucleotides cannot be added w/o a template, DNA polymerase only extends existing polynucleotides (requires primers)

where does DNA synthesis initiate in bacterial DNA replication

a single site on the chromosome called the origin of replication (oriC),

how does synthesis elongate in a bacterial chromosome

via bidirectional replication forks around the bacterial chromosome

where do the bidirectional replication forks terminate replication in bacterial chromosome

at the opposite side from the origin of replication (oriC)

prokaryotes have what kinds of chromosomes

one main single, large, circular DNA molecule and accessory parts of genome made of smaller bits of DNA called plasmids

do plasmids have an origin of replication

yes

how is DNA replication initiated

by the binding of DnaA proteins to the DnaA box sequences

what does the DnaA proteins binding to the DnaA box sequences stimulate

the cooperative binding of an additional 20-40 DnaA proteins to form a large complex —> DnaA proteins binds to the DnaA box sequences and then bind to each other

what follows the binding of DnaA proteins to the DnaA box sequences and those proteins bind to each other

the region wraps around the DnaA proteins and separates the helix in the A/T rich region

what does wrapping of DnaA proteins do to the DNA

creates a strain on the DNA enough to overcome the weak A/T pairing and create small sections of single stranded DNA and initiates replication

helicase (DnaB)

enzyme that catalyzes the denaturing of double stranded DNA into single stranded DNA

replisome

the DNA polymerase, primase, and accessory proteins needed to carry the process of replication

when is initiation of replication over

when there are two replisomes on the DNA

what DNA polymerase is involved in DNA repair

DNA poly II

what DNA polymerase is part of the replisome

DNA poly III

primer

piece of nucleic acid needed for DNA polymerase to extend adding nucleotide residues to free 3’ end

what polymerase can make DNA de novo and which cannot

RNA polymerase can make DNA de novo and DNA polymerase cannot make DNA de novo

what is required for elongation of replication to start

short RNA sequence of nucleotide residues made by enzyme primase

during replication elongation you see what components

helicase (@ each rep. fork), primase (puts together two nucleotides and adds additional nucleotides to the 3’ end), and DNA polymerase (adds nucleotides to the 3’ end after primase is done)

what enzyme continues the process of replication elongation after primase makes the short RNA primer and adds nucleotides to the free 3’ end

DNA Polymerase

where is there a replisome

at either end of the replication bubble

as long as a single strand of DNA is available, ____ will “lay down” primers for what strand

primase, on either template strand (cause there’s 2 when you unwind DNA) can receive primers to continue synthesizing the leading and lagging strand

leading strand and direction of synthesis about replication fork

continuously synthesized strand synthesizing toward the replication fork

lagging strand

discontinuously synthesized strand synthesizing away from the replication fork

activity of DNA helicase

break the hydrogen bonds between the two strands of the parental duplex

activity of DNA Primase

lay down new primers in the 5’ to 3’ direction

activity of DNA polymerase

bind to the 3’ ends of the primers laid down by primase and add nucleotides to the 3’ end to synthesize new DNA

where is the most recent okazaki fragment located in reference to the replication fork

most proximal (closest)

what are the spaces between okazaki fragments and how can they be resolved

missing phosphodiester linkages that are resolved by DNA ligase

activity of DNA polymerase 1

removes RNA at 5’ end of neighboring fragment and fills gap (space between Okazaki fragments)

DNA Polymerase I removes RNA primers using enzymatic activity known as

exonuclease activity

exonuclease activity

removing nucleotides

what kind of exonuclease activity (what direction) does DNA Polymerase I have

both 3’ to 5’ and 5’ to 3’

DNA Ligase activity

adds phosphodiester linkage, phosphate comes from ATP (energy required to make bond)

single-stranded binding protein

keeps the parental strands apart and stabilizes the strands so the bases don’t rebind

what relieves supercoiling as the DNA is being unwound by helicase

topoisomerase

after elongating the first okazaki fragment what does DNA Pol. III do?

dissociates and reattaches to the next RNA primer and repeats this process each time DNA Pol. III bumps into an RNA primer

DNA Gyrase is what kind of enzyme and does what

a type of topoisomerase that removes extra twists/relieves supercoiling by cutting the DNA allowing it to rotate and then rejoins the DNA strands

what are ‘ter’ sequences and where are they located

termination sequences T1 and T2, that are opposite the oriC (origin of replication)

how is the movement of the replication forks stopped

the protein ‘tus’ (termination utilization substance) binds to the ter sequences

T1 ter sequence allows and prevents the movement of which replication forks

Allows advancement of CW-moving fork, prevents CWW-moving fork

T2 ter sequence allows and prevents the movement of which replication forks

Allows advancement of CWW-moving fork, prevents CW-moving fork

difference between eukaryotic and prokaryotic chromosomes

eukaryotes’ chromosomes are longer, linear, and there are multiple of them that require multiple oriCs as opposed to prokaryotes that have a single, circular molecule & a few small accessory molecules i.e. plasmids)

origin recognition complex (ORC) and what type of cells (euk or prok) do they reside in?

complex of proteins in eukaryotic cells that recruits other pieces to denatur the DNA in A/T rich region

what is the main difference between prokaryotic and eukaryotic replication and how is that difference resolved

the amount of DNA that needs to be copied, resolved by multiple origins of replication in eukaryotes

what potentially occurs after the most recent okazaki fragment primer naturally degrades

(there’s no free 3’ end b/c okazaki fragments synthesize 5’ to 3’ so you’re left with a 5’ end and) overhang potentially occurs from the template strand

problem of 3’ overhang of DNA template strand and solution

losing genetic material every time you go through a replication, solution is telomeres

telomere

disposable repeating sequence at ends of chromosomes *through cell division, telomeres will shorten over time (a way a cell ages)

telomerase activity

enzymatic activity that lengthens telomeres in 5’ to 3’ direction (does not occur in every cell type i.e only in gamete producing cells in humans)

telomerase process to correct overhang of template DNA so newly synthesized DNA can match

1. telomerase RNA base pairs with DNA template

2. telomere extension occurs (add DNA nucleotides to 3’ end using the embedded telomeric RNA as a template i.e RNA directed DNA polymerase)

3. telomerase translocated to extended 3’ end (releases from new extension and reanneals to the end for continued elongation

4. telomerase extends 3’ end of telomere (because using same template sequence every time the DNA sequence will be the same repeated over again)