Cell Biology Exam 2 Theisen

Replication Fork

place where strands are unwinding and opening. DNA is unwound in region to be replicated. There are two synthesis sites at each fork, and 4 in total. Each strand serves as a template for synthesis of a new strand.

DNA replication is

semiconservative

Separation of DNA strands is

unfavorable, because a double helix is stable. this is accomplished through coupling reactions.

Helicase continuously binds and hydrolyzes

ATP molecules, conformational changes cause it to screw onto one strand, pries two strands apart as it moves.

Single strand binding proteins

bind to open single-strand portions of DNA, prevent self-repairing or re-annealing without blocking access to bases.

DNA is synthesized in what direction?

5' to 3' direction ONLY, because the energy is carried on the incoming nucleotide.

Hairpins

single strand intrastrand base pairing

DNA primase

required to initiate replication, synthesizes initial primer strand. Strand made from rNTP's, resulting in RNA primer attached to the DNA template.

The 3' to 5' strand is called the

leading strand

On the leading strand the process of priming with RNA and replacing with DNA

happens only at the start of each replication fork.

-Once for prokaryotes

-Multiple times for eukaryotes.

DNA template and complementary strands are

antiparallel

Lagging Strand

Replication of the 5' to 3' template involves movement of the replication machinery away from the replication fork. this maintains the 5'-3' direction required for all new strand synthesis.

On the lagging strand the process of priming with RNA and DNA replacement is

continuous, replication machinery must return to the replication fork.

Okazaki fragments

Small fragments of DNA produced on the lagging strand during DNA replication, joined later by enzyme DNA ligase to form a complete strand. The lagging strand is synthesized in fragments.

Using RNA instead of DNA requires

replacement

**Ligase needs an enzyme

they need a source of energy (ATP molecule). *Review*

Sliding ring protein

DNA polymerase does not associate directly with DNA. This protein keeps polymerase close to DNA enable rapid movement and facilitates dissociation when previous Okazaki fragments are encountered.

ATPase

enzyme that catalyzes hydrolysis of ATP. (Ligases, Helices, etc.) coupling and unfavorable reaction to a favorable reaction.

Topoisomerase

as helical dan unwinds it rotate, DNA cannot rotate easily due to space limitations, this twisting can cause tangling, topoiomerase solves this.

Topoisomerase I

hydrolyzes the breaking of the phosphodiester bond. captures some of the energy released to repair the phosphodiester bond previously broken. Mechanical energy is a large released of energy.

Topoisomerase II

An enzyme that breaks a DNA double helix, rotates the ends, and seals the break.

DNA replication requires

- topoisomerases

- Deoxyribonucleoside Triphosphate

- Ribonucleic Triphosphates

Occasional changes to DNA sequences

benefit a species over the longterm.

Mutation

change in the DNA sequence, a failure in the repair mechanisms.

Rate at which mutations occur

Mutation rate

Damage in the DNA

is not a mutation

Mutations are

extremely rare, 1 base change per / 10^9 (a billion) / replication

Range from bacteria to mammals.

Most mutations are

"silent"—effect is not observed in the phenotype

Mutation is not

in a protein coding region (intron)

Lethal loss of function

non-viable organism, nothing to observe.

Replication mistakes

far exceed the rate of mutation

Proofreading mechanisms

operate during replication to identify and correct error.

Two primary mechanisms of Proofreading

-DNA polymerase

-Exonucleolytic proofreading

Imperfect base pairing involving "wrong" base leads to

random conformational change. no bond formation, base dissociates.

Tautomeric forms

alternate chemical forms of nucleotides that allow pairing with non complementary bases.

5' to 3' facilitates proofreading

when exonucleolytic proofreading strips away a mis-paired base, the energy for the next phosphodiester bond is not also stripped away.

exonucleolytic proofreading

Operates if an incorrect base is accidentally covalently bonded by removing it out of the strand.

It is theorized that DNA synthesis occurs only in the 5' to 3' direction because

bond energy for correcting errors is always available via incoming nucleotide.

Lagging strand

Replication of the 5' to 3' template involves movement of the replication machinery away from the replication fork.

Chromatin Assembly Factors (CAFs)

aid in histone assembly (histone chaperones)

New DNA inherits combination of old and newly synthesized

histones, pattern of histone modification is inherited.

Two subunits of Chromatin Remodeling Complexes

Code reader subunits: binds to previously modified histones.

Code writer subunits: Modifies adjacent histones.

Reader-writer complex

parental nucleosomes with modified histones, only half of the daughter nucleosomes have modified histones, parental pattern of histone modification re-established by read-writer complexes that recognize the same modifications they catalyze.

Synthesis of lagging strand cannot occur at

the end of a DNA molecule, no "upstream" strand for DNA polymerase-a to bind to and add RNA primer. DNA molecules would become shorter with each replication.

Telomerase

multi-subunit ribonucleoprotein complex assembled in the nucleolus, binds to the telomeric region, uses a built in RNA template elongation of parental "lagging" strand in the 5' to 3' direction.

(Artificial extension allows DNA to bind upstream).

In telomeres, the 3' strand of DNA from the lagging strand is

always longer, this looks like broken DNA to cell repair machinery, telomeres distinguishes itself from broken DNA by having the longer strand fold onto itself.

Telomere Length

- regulated by cells and organism

- synthesis of telomerase "turned off" of "slowed down" in certain cells

- after many generations or if chromosomes become defective

- Cell dies-Replicative cell senescence

- certain cancers derived from flaws in this mechanism.

DNA damaging events

thermal fluctuations (extremely common), metabolic "accidents" (unintended oxidations), rearrangements caused by radiation (UV, X-ray, etc.), environmental toxins (mutagens, carcinogens)

DNA Damaging events are NOT

Replication errors

Almost all DNA damage is identified and repaired

cells synthesize many different kinds of protein, most repair involves use of template as a guide for the repair.

DNA repair mechanisms

-Base Excision Repair

-Nucleotide Excision Repair

-Double Strand Break Repair

DNA glycosylase

slide along DNA and separate bases from their complement. "Flipping Out". Glycosylase has 6 different types corresponding to the amount of base pairs. Each glycosylase recognizes its own specific altered base. When an altered base is recognized, a conformational change occurs which activates enzymatic activity. Base is removed from sugar via hydrolytic reaction.

Glycolase removes altered base from sugar via

hydrolytic reaction, AP endonucleoase and AP phosphodiesterase them cut a remove the remaining backbone.

AP endonuclease (AP = Apurinic or pyrimidinic) and Phosphodiesterase

in Base Excision Repair, the enzymes that cut one side of the bond.

DNA polymerase

replaces the removed base in Base-Excision Repair. (complete repair)

DNA ligase

reforms the bond on either side in Base-Excision repair. (complete repair). Receives energy from ATP.

Deamination

"unnatural" base, each unnatural base has its own glycosylase. DNA evolved from RNA, evolution selected Thymine (T) over Uracil (U). Deaminated C = U, every DNA U = error.

Artificial extension allows

DNA polymerase to bind upstream

Replication cell senescence

cell dies, how certain cancers have arisen.

Phosphodiester bond

relatively low energy bond, therefore relatively stable

DNA is stable but is NOT

indestructible

Most mutations:

are silent, to be considered a mutation it must be passed onto the next generation (must be heritable).

ATP hydrolysis

favorable reaction

Phosphodiester bond formation

unfavorable reaction

When Cytosine is deaminated

Uracil is formed

Pyrimidine dimer

Structure in which a bond forms between two adjacent pyrimidine molecules on the same strand of DNA; disrupts normal hydrogen bonding between complementary bases and distorts the normal configuration of the DNA molecule

nucleotide excision repair

uses acid-base reactions

Non-homologous end joining

broken ends simply brought together and rejoined. results in apparently acceptable mutation,

Homologous recombination

a mechanism that repairs DNA double stranded breaks by preserving original sequence. requires sister chromatid and can only operate after replication (S and G2 phases).

Homologous recombination (general recombination)

involves interactions between homologous sites on two different but homologous chromosomes.

Transposition & Site Specific Recombination

mobile genetic elements, involve interactions between non-homologous sites, different sites on the same chromosome.

Homologous Recombination exchanges genetic material by

crossing over during meiosis. this is favorable evolutionarily.

How is the single stranded piece of DNA generated?

*strand breakage event—already illustrated

*enzymatic breakage and partial digestion (first step in meiotic "crossing over")

Steps of homologous recombination

1. Part of DNA molecule becomes single stranded.

-single-stranded binding proteins maintain single strand.

2. Single strand "invades" a homologous double stranded DNA molecule.

-Base pairs with complementary region of DNA, displacing the original partner.

-Heteroduplex is formed

How is the single strand able to find and pair with its complement in a different DNA molecule?

-mechanism mediated by special proteins which facilitate a strand invasion.

-E. coli protein - RecA

-Eukaryotic protein - RAD 51

Strand Invasion: RecA/Rad51 protein

multimeric, filamentous proteins, which have monomers that posses DNA binding sites that bind to single-stranded invading DNA. Slide across homologous target and identify complementary region on target DNA by unknown mechanism that binds simultaneously to both DNA molecules. Complete strand invasion results in heteroduplex.

In double stranded break repair

DNA polymerase forces unidirectional branch migration.

Meiotic Recombination

begins with a targeted double stranded break. Enzymes go in and specifically target a DNA molecule.

Holiday Junction

intertwined structure that forms during the process of genetic recombination, when two double-stranded DNA molecules become separated into four strands in order to exchange segments of genetic information.

(intertwined/completely non-functional structure) usually between sister chromatids one maternal and one paternal.

In humans only ___ of strand resolution result in crossover.

10%

Nonreciprocal resolution of Holiday junction

results in unequal contribution of allele from one parent to offspring. Gene conversions are rare and silent.

Mobile genetic elements:

100's to 10,000's bp long, Move form one region of the genome (donor) to another region (target), May move with same chromosome or between chromosomes, some elements can move between organisms.

Many inactive MGE's (Mobile Genetic Elements)

remain fixed in genomes (largest component of human DNA = 45%. Often alter neighboring sequences at the target site.

Two broad classes of movement

1. transposition

aka transposable elements (transposons), no homology required between sites, DNA-only transposons, retrovirus-like retrotransposons and retroviruses.

2. conservative site specific

common in prokaryotes, limited homology between extrachromosomal DNA (bacteriophages and plasmids).

Reverse transcriptase

catalyzes synthesis of new double-stranded DNA molecule from its own mRNA molecule.

Integrase

catalyzes insertion of newly synthesized DNA elsewhere in host genome.

Reverse Transcriptase and Integrate are referred to as a __________ mechanism

Copy and Paste

Retroviral-like retrotransposons

have mechanisms similar to retroviruses, no protein coat and cannot leave the cell, it is not a true retrovirus.

True Retroviruses

- Same mechanism as retroviral-like

*however, mRNA also codes for coat proteins, have a protein coat allows movement of mobile genetic element

between cells and organisms.

Evolutionarily related processes

- DNA repair

- homologous recombination

- site-specific recombination

- all involve

- site recognition

- cutting/and or excision of nucleotides

- insertion and replication of new sequence

- all make use of endonucleases, exonucleases, phosphodiesterase, helicase, polymerase, ligase, SSB's, etc.

All organisms have

mRNA, rRNA and tRNA

Archaea and Eucaryotes

snRNA and snoRNA

Eukaryotes have

siRNA, miRNA, piRNA, IncRNA

Bacteria and Archaea

crRNA(CRISPR -clustered regularly interspaced, short palindromic repeats).

A copied portion of DNA is called

a gene

RNA

ribose, uracil, single-stranded. single strand allows tertiary structure.

Eukaryotic RAD51 and prokaryotic RecA are homologous. Homologous proteins:

look and/or function alike because they are descended from a common ancestor.

Initiation of prokaryotic transcription

ơ (sigma factor) factor binds to the polymerase, changes the shape of the polymerase and allows it to associate with DNA, until reaching the promoter, the sigma factor (ơ) then binds to the promoter, a conformational change occurs (energetically favorable this is the source of energy), opens a double helix, ơ factor dissociates, elongation proceeds.

The complex slides along DNA until

it encounters a promoter

After ơ dissociates

jaw-like structure forms that holds the DNA in place.