DNA Replication

Nucleoside

Includes a sugar and a nitrogenous base

Function in cell signaling and a precursor for genetic material

Nucleoside Bases

Adenosine, Guanosine, Cytidine, and Thymidine

What base pair needs more energy to break and why?

C to G pair because it has 3 hydrogen bonds instead of 2

Major Groove

The wide, prominent indentation in the double helix

Minor Groove

The small, narrow indentation in the double helix

What type of bonds connect the base pairs?

Hydrogen Bonds

What bonds connect the base pairs to the sugar-phosphate backbone?

Phosphodiester bonds

Phosphodiester bonds

The bonds that connect the nucleotides to the backbone; Formed by condensation reactions between the phosphate and sugar-hydroxyl groups

Polarity

The way in which nucleotide strands line together gives them biological polarity which means they run in antiparallel to each other

Purines

The double ringed bases of DNA

What bases are purines?

Guanine and Adenine

Pyrimidines

The single ringed bases of DNA

What bases are pyrimidines?

cytosine and thymine

What type of reaction links the nucleotides together?

Dehydration reaction which is catalyzed by DNA polymerase

What type of polymerase is used in replication?

DNA polymerase

What type of polymerase is used in transcription?

RNA polymerase

What is the main organelle used in translation?

ribosomes

Replication machine

clusters of proteins that include replisomes that help copy DNA

Semiconservative Model

Each parental strand serves as the template for one new strand. Each daughter DNA double helix is composed of one of the original strands plus one strand that is completely new.

The Centrifugation in what compound allowed what process to occur?

the centrifugation in a cesium chloride gradient allows the separation of heavy and light DNA

Meselson-Stahl Experiment Part 1

Bacteria grown in 2 mediums (N15 and N14) loaded and isolated into different tubes to centrifuge. Based on the density, DNA separates in different ways.

Describe the pattern of separation for more dense DNA.

Heavy N15-DNA forms a high density band, closer to the BOTTOM of the tube

Describe the pattern of separation for less dense DNA.

Light N14-DNA forms a low-density band, closer to the TOP of the tube

What did scientists find after the first part of the experiment?

They found that the marker was in the middle, which meant that DNA was either made from the dispersive model or the semiconservative model

How did they differentiate between the dispersive model and the semiconservative model?

They denatured the DNA from each type of sample which makes the DNA split. When it splits, the markers from the bacteria show up.

What was the final result of the experiment?

It showed 2 bands (1 of each type of density) instead of a single thick band with the same density which demonstrated a semiconservative model of DNA

Replication Initiator Proteins

They recognize sequences of DNA at the replication origins and locally pry apart the two strands of the double helix

The exposed single strands during replication serve as what?

Templates for copying the DNA

What type of base pair connections do the replication origins have?

usually have more A-T connections since they are easier to open due to less bonds

1?

Template DNA

2?

Newly synthesized DNA

3?

Replication forks

4?

Replication origin

How many replication origins are there in bacteria?

1

Replication happens in ____directions from the origin

both

Nucleosomes

Structures made of DNA and the protein complexes around which the DNA is wrapped

Chromatin

a mixture of DNA and proteins that form the chromosomes found in the cells of humans and other higher organisms

Histones

package the massive amount of DNA in a genome into a highly compact form that can fir in the cell nucleus

Chromatin packaging method is found in what type of cells?

Eukaryotic cells

What direction does DNA synthesis occur?

5’ → 3’

What end is each nucleotide added to on the growing new strand?

3’ end due to the free hydroxyl group

DNA Polymerase

a multiple protein sub-unit complex that adds a deoxyribonucleotide to the 3’ end of a growing DNA chain

What happens to the triphosphate group on the incoming nucleotide when added to the chain?

broken into pyrophosphate and then further into 2 single inorganic phosphates

What is the purpose of breaking the pyrophosphate?

it prevents a reversible reaction meaning it prevents it from gaining back the third phosphate so that the nucleotide can stay in place

When you break down the phosphates what is actually happening?

energy is being released

Why is DNA polymerase also a catalyst?

it catalyzes covalent linkage of nucleotides into the growing new strand

Polymerization reaction

involves the formation of a phosphodiester bond between the 3’ end of the growing DNA chain and the 5’-phosphate group of the incoming nucleotide

The nucleotide incoming to the polymerization reaction in the formation of DNA enters the reaction as what molecule?

deoxyribonucleoside triphosphate

The energy for polymerization comes from where?

the deoxyribonucleoside triphosphate; hydrolysis of the high-energy phosphate bonds fuels the reaction that links the nucleotide monomer to the releasing pyrophosphate

Does DNA polymerase dissociate?

No, it stays associated with the DNA and moves along the template strand stepwise for many cycles of the polymerization reaction

At the _____ _____, the newly synthesized DNA strands are of ____ ____

replication fork, opposite polarities

Okazaki Fragments

Since both strands are synthesized in the 5’→3’ direction, the lagging strand of dNA must be made initially in a series of short DNA strands

What mechanism does DNA polymerase use to replicate the lagging strand?

backstitching

Describe the movement of DNA polymerase as it synthesizes the lagging strand

synthesizes the Okazaki fragments in the 5’→3’ direction and then moves back along the template strand (toward the fork) before synthesizing the next fragment

What strand takes longer to synthesize and why?

The lagging strand takes longer because there are more steps and it takes more time

In what direction is the leading strand copied?

towards the origin of replication

What happens if an incorrect nucleotide is added to a growing strand?

the DNA polymerase will cleave it from the strand and replace it with the correct nucleotide before continuing

Proofreading

correcting a rare mistake; carried out by a nuclease that cleaves the phosphodiester backbone

The proof-reading nuclease works on what end of the DNA strand?

the free end

Exo-nuclease activity

when the nuclease works on the free end of the DNA strand

Endo-nuclease activity

works on the middle end of the strand

What direction does proofreading occur?

3’→5’

DNA polymerase contains _____ ____ for synthesis and proofreading

separate sites

What does the p side do?

polymerization activity

What does the E side do?

proofreading activity (editing)

What happens when the polymerase adds the incorrect nucleotide?

the newly synthesized DNA strand transiently unpairs from the template strand and its growing 3’ end moves to the error-correcting catalytic site (E) to be removed

What would happen in terms of proofreading if DNA was copied 3’→5’ instead of 5’→3’?

Polymerization can’t proceed because there is no high energy bond (phosphate) available to drive the reaction

What are RNA primers synthesized by?

primase (a type of RNA polymerase)

What is a key difference between DNA polymerase and primase?

unlike DNAP, primase can start a new polynucleotide chain by joining together two nucleoside triphosphates without the need for a base paired 3’ end as a starting point

DNA polymerase is _____ dependent

primer

the short length of RNA base pairs from the primer serve as a ____ for DNAP

starting point

What would happen if there wasn’t a primase?

there would be no initiation of DNA synthesis on either strand and no Okazaki fragments would be made

How are primers removed?

by nucleases that recognize an RNA strand in an RNA/DNA helix and degrade it

What happens when the RNA primers are removed?

it leaves a gaps that are filled in by a repair DNA polymerase that can proofread as it fills in the gaps

What is the final step after removing the primers?

the Okazaki fragments are finally joined together by an enzyme called DNA ligase

How exactly does DNA ligase connect the fragments?

DNA ligase catalyzes the formation of a phosphodiester bond between the 3’-OH end of one fragment and the 5’-phosphate end of the next, thus linking up the sugar-phosphate backbones

What energy molecule is required for the linking of the backbone through ligase?

ATP

On what strands do you need ligase for?

both strands

Protein clamps

DNA polymerases are held on the leading and lagging strands by these that allow the polymerases to slide

When does the clamp detach and on what strand?

detaches each time the polymerase completes an Okazaki fragment on the lagging-strand template

Clamp loader

required to attach a sliding clamp each time a new Okazaki fragment is begun

DNA helicase

At the head of the fork, it unwinds the strands of the parental DNA double helix

Single-strand DNA binding proetins

they keep the DNA strands apart to provide access for the primase and the polymerase (almost like a reverse magnet between the two DNA strands)

DNA topisomerases

relieve the tension that builds up in front of a replication fork

Why does tension build up in the DNA?

there is tension in the overwound DNA because the chromosome is too large to rotate fast enough to relieve the buildup of torsional stress

How do the topoisomerases relieve the stress?

they generate temporary nicks in the DNA, once the tension is released, it reseals the nick before falling off

Without a special mechanism to replicate the ends of linear chromosomes, what would happen to the DNA?

DNA would be lost during each round of cell division

Why can’t the ends of the lagging strand be completed normally?

because once the final RNA primer has been removed there is no way to replace it with DNA

Why must the gaps created on the lagging strand be filled?

to keep the chromosome ends from shrinking during cell division

Telomeres

the end of a chromosome marked by several repeated sequences

Reverse transcriptase

responsible for the RNA template used to help synthesize more DNA repeats at the end of the chromosome

What do cancer cells have in relation to telomeres?

they make more telomerase to protect themselves since they are rapidly dividing