Micro Lecture 14 - Molecular Info Flow: Replication

Information is…

independent of the medium upon which it is stored or encoded

Meischer

1869

Nuclein

Griffith

1928

Transformation

Avery, Macleod, McCarty

1944

transformation

Hershey and Chase

1944

Blender experiment

Chargaff

1948

the “rules” - took things apart to discover A=T and G=C

What are monomers

Bases

Nucleosides

Nucleotides

What are bases

A C G T

What are nucleotides made of

base

sugar

phosphate

What are nucleosides made of

base

sugar

Know numbers on monomer slide (1,2,3..C)

…

What are purines and their structure

A and G

two ring structure

Larger

What are pyrimidines and their structure

C, U, and T

one ring structure

smaller

what did Franklin and Wilkins do

did X-ray diffraction pattern from a DNA smear

knew DNA was a helix but didnt recognize what they were looking at

Watson and Crick

figured out DNA structure from X-ray

Watson and Crick’s model

(how many nm per twist)

(how many base pairs per twist)

(how many H bonds between different base pairs)

postulated anti-parallel double stranded molecule

bases inside

3.4 nm per twist

10 bp per twist

C - G (3 H bonds)

A - T (2 H bonds)

explain Chargaff’s rules as they were understood

Purines match with pyrimidines

hydrogen bonds form between them

both DNA strands have the….

same amount of information

bases in one strand are___________ to those in the other strand

complementary

What is the modern central dogma (1950-today)

DNA —> RNA —> protein

write out todays chart

What did Meselson and Stahl observe

in 14N media, there was evidence of semiconservative DNA replication

What is the genome

complete cell DNA sequence

what is the genotype

specific DNA sequence

what is phenotype

appearance and/or behavior

genotype + environment = phenotype

the genome consist of ______ molecules (free-living cells range from _________ nucleotides)

Large

583,000 up to 150,000,000,000

Genomes of prokaryotes are _______ and ________ (mostly)

circular

haploid

DNA is ______ to fit in the cell

packed

the ______ of an E. coli cell is packed

nucleoid

Circle of ___________ is __________ the size of the cell

ds DNA

1500x

multiple loops held by…

each loop has _____________

anchoring proteins

supercoiled DNA…

What effect does supercoiling have on DNA

it compacts it

positive supercoils are

over winding

Unsupercoiled DNA has ____ wind(s) for _____ bases

1 wind for 10 bases

negative supercoils…

under winding

what do supercoils do to DNA

it twists DNA

What do topoisomerases do

they regulate supercoils

What does Type I Topoisomerases do

they relieve torsional stress caused by supercoils

what do Type II Topoisomerases do

they introduce negative supercoils

What do archaeal topoisomerases do

They introduce positive supercoils

DNA replication is ___________

semiconservative

Where does DNA replication begin

at oriC

DNA is opened at ______

oriC

What are the general steps of DNA replication

DNA opened at oriC

polymerization occurs bi-directionally around chromosome

Helicase unwinds DNA and recruits Primase

Primase begins replication

DNA polymerase III clamped to strand

DNAP III adds nucleotides in 5’ —> 3’ direction

RNase H removes primers

DNAP I fills gaps

Ligase seals in Okazaki fragments

What follows the opening of DNA at oriC

polymerization

DNA polymerization moves…

bi-directionally around the chromosome

____________ melts DNA

DNA Helicase

What places helicase and where is it placed

loader places helicase at each end of origin and each move in opposite directions

What does DNA helicase do

it unwinds DNA

Helicase recruits _____

primase

what does DNA polymerase need

a free 3’OH

What is primase’s function

to begin replication

builds an RNA primer forms 3’ OH for DNA to attach

primer recruits…

clamp loader to each strand

What does the clamp (recruited by primer) bind to strand

clamp binds DNA Polymerase III to strand

Which way does Polymerase move down the strand

in the 5’ → 3’ direction on each strand (add bases to the 3’ end)

where does energy for polymerization come from

from phosphate groups on the recently added nucleotide

each fork has…

two strands

what is the leading strand

steady growth occurs here

the leading strand follows helicase

what is the lagging strand

polymerase continues to previous primer and the clamp loader places primase on new site (the primase adds a short RNA primer, which is continued by the polymerase to form an okazaki fragment)

it is built in pieces because it cannot continually add bases because it is in the 3’ —> 5’ direction

the lagging strand has ___________________

okazaki fragments

DNA present in _________ base pieces

1000

What does RNase H do

it removes primers

How many primers are there for each leading strand

One primer

How many primers are there for each lagging strand

many

how many primers are there per Okazaki fragment

one

Gaps (segments of Okazaki fragments) are created by _________________

DNA Polymerase I

what seals the nicks in DNA (the Okazaki fragments)

Ligase

Both forks move to _______ Sites

ter

Fork movement…

is simultaneous

move in opposite directions until both meet again at terminus

Replisomes are…

actually stationary

(enzymes involved in DNA replication for the replisome)

DNA is threaded through…

the replisomes

What are plasmids

extrachromosomal pieces of DNA

_______________________ plasmids

low-copy-number

How many plasmid copies are there per cell for Low-copy-number plasmids

one or two

_______________________ plasmids

High-copy-number

How many plasmid copies are there per cell for high-copy-number plasmids

up to 500 copies per cell

what do high-copy-number plasmids do

they divide continuously

they randomly segregate

What are the two types of plasmid replication

bidirectional and unidirectional

bidirectional replication of plasmids is…

similar to chromosomal replication

unidirectional replication of plasmids is also called

rolling circle replication

Unidirectional replication starts at…

nick bound by RepA protein

unidirectional replication…

provides 3’ OH for replication

Helicase moves around plasmid repeatedly

unidirectional replication is used by many _______________

bacteriophage viruses

plasmid genes are…

advantageous under special conditions

what are some advantages to plasmid genes

antibiotic resistant genes

genes encoding resistant to toxic metals

genes encoding proteins to metabolize rare food sources

virulence genes to allow pathogenesis

genes to allow symbiosis