BIO 221: DNA structure and Replication Enzymes

What are the 4 properties of genetic material?

Information, replication, transmission, and variation

Information (genetic info)

must contain info needed to construct an entire organism

Replication (genetic info)

must be able to accurately copy the genetic info

Transmission (gentic info)

must be able to pass replicated genetic info to daughter cells during division

Variation (genetic info)

diffrences in genetic info must account for variation within and between species

How is information stored in DNA?

In the sequence of nucleotides

Frederick Griffith experiment

he injected mice with strains of streptococcus pneumonia to see the differing effects it would have, proving DNA is genetic material

bacterial species used in Griffeth’s experiment

Streptococcus pneumoniae

Two strains of streptococcus pneumonia

S strain (has capsule) and R strain (no capsule)

S strain of s. pneumoniae

produces thick, gooey capsule making the bacteria virulent

R strain of s. pneumoniae

doesn’t produce capsule, non virulent

Competence

the ability to take up exogenous DNA from environment

Competence of denatured S strain

allows bacteria to express/exhibit new traits

Effect of S strain on mouse

killed the mouse because its virulent

Effect of R strain on mouse

did not kill the mouse because its non-virulent

Effect of denatured S strain + R strain

denatured S strain transformed the R strain giving it a capsule killing the mouse because its now virulent

What do you expect would have happened if the S type

cells were not heat killed when mixed with the R type cells

in the 4th injection?

If the cells didn’t die then the DNA wouldn’t transfer so the result would be the same (the mouse would die)

Why is it that the S type cells kill the mouse and R type cells don't?

The S type have a thick capsule where the mouses immune system can’t target, but R type have a thin slime layer so that it is targeted and killed

What is the Avery, MacLeod, & McCarty Experiment

purified DNA, RNA, proteins (enzymes), and other materials from heat-killed S-type and mixed them with R-type to see which ones could transform living R-types to S-types.

Building blocks of nucleic acids

nucleotides joined by phosphodiester bonds

How are nucleotides linked to form strands?

phosphates join to 5' while OH join to 3' creating a chain held together by hydrogen bonds

Basic structure of DNA

double helix held together by hydrogen bonds between base pairs

What are the key features of the DNA double helix?

The 2 strands for a double helix, antiparallel

Bases in each strand hydrogen bond according to

the AT/CG rule

C-G pairs have 3 H-bonds, A-T pairs have 2 H-

bonds

For each full turn, there are ~10 nucleotide pairs

Basic structure of a chromosome

very long double stranded molecule with a histone in the center

Histone

help give organization and structure to chromosome, can be modified to influence gene expression

Genome

the entirity of a cells genetic info

Components of nucleotides

phosphate, 5 carbon ring structure, nitrogenous base

Phosphate attached to nucleotides are on

Carbon 5 (DNA and RNA)

DNA base pairs

A to T and G to C (on Carbon 1)

RNA base pairs

A to U and G to C (on Carbon 1)

Purines

double ring; a,g

Pyrimidines

single ring; t, c, u

Importance of 5' and 3' ends

they dictate the directionally of DNA synthesis (5' to 3' synthesis)

Types of bonds in DNA

double (A-T) and triple (G-C) hydrogen bonds

Orentation of DNA

anti-parallel

Characterictics of DNA replication

Semiconservative, Bidrectional, 5' to 3' directions, DNA polymerase

Semiconservative replication

two strands of DNA are separated, synthesizes a new complementary strand 5’ to 3’ direction, resulting in two identical DNA strands; one is old (template) and new

Bidirectional replication

starts at origin of replication and creates a replication bubble

DNA synthesis directionality

5' to 3'

Which direction is a template sstrand READ

3’ to 5’

What enzyme synthesizes DNA during replication

DNA polymerase

DNA polymerase

enzyme which synthesizes the new DNA strand; requires a template strand and a free 3' OH

What are the requirements for DNA polymerase synthesis

template strand and free 3’ OH group

melting of DNA

breaking hydrogen bonds that hold two strands of DNA together

replication bubble

area where replication occurs, containing two replication forks

proteins required for DNA replication

DNA helicase, DNA topoisomerase, and ssDNA binding proteins

DNA helicase

binds and melts DNA at replication forks, separating dsDNA into ssDNA templates

DNA topoisomerase

prevents supercoiling by breaking phosphodiester bonds and bonding them back together

Single-stranded DNA binding proteins

bind to ssDNA templates to prevent them binding back together (annealing)

What happens when DNA is supercoiled

It can’t unstrand/replicate DNA

Activity of DNA polymerase

read 3’ to 5’ and synthesizes DNA strands from dNTPs bonding them together with phosphodiester bonds

dNTPs

deoxynucleotide triphosphates (dTTP, dCTP, dATP, dGTP)

Structure of DNA polymerase III

core subunits, tau subunits, and gamma complex

Core subunits of Poly III

alpha, epsilon, and theta

alpha subunits

synthesize new DNA strand

Epsilon subunit

proofreads and corrects errors made by alpha subunit

theta subunit

assists epsilon in proofreading

Tau subunit of PolyIII

connects the core subunit to the gamma complex

Gamma complex of PolyIII

loads and unlods the beta clamp which is holding dNTPs onto the DNA strand

Primase

synthesizes RNA primers that are complementary to both templates at origin

Primasome

primase + helicase essential for initiating replication

You are studying a mutant strain of bacteria and observe that after

replication is complete, the bacterial chromosome consists of mostly DNA

with short stretches of RNA present throughout the chromosome. A

mutation in which of the following would be most likely to cause this?

DNA Polymerase I because DNA polymerase I removes the RNA primers and replaces them with DNA. If this enzyme wasn't functioning properly, the RNA primers would not be removed.