DNA Replication and Damage Repair

Without accurate replication, the genetic material of…

resulting cells would be riddled with errors

mitosis

the process of nuclear division

cytokinesis

the division of the cytoplasm

sister chromatids

chromosomes that have duplicated have two of these

Interphase

where cells spend most of their time, in between divisions

M phase

the events of mitosis

What happens during interphase?

the amount of nuclear DNA doubles during the S phase

G1 phase separates

S phase from the previous M phase

G2 phase separates

S phase from the next M phase

The Watson and Crick model of DNA structure suggested…

a mechanism for how the base-paired structure could duplicate itself

semiconservative replication

two strands of each new DNA molecule was derived from the parent molecule and the other strand was newly synthesized

Meselson and Stahl with Vinograd showed that…

bacterial replication is semiconservative by using N-14 and N-15 to distinguish newly formed DNA strands from old

Taylor, Woods, and Hughes completed an experiment with

a eukaryotic organism using ³H-thymidine and autoradiography

Replication forks

formed where replication begins and then proceeds in bidirectional fashion away from the origin

origin of replication

the site where DNA replication initiates, synthesis is initiated by groups of initiator proteins

The sequence of DNA replication initiation is

AT rich and about. 245 bp in length

consensus sequences

the sequence varies among bacterial species but contains recognizable, similar sequences, this is what those sequences are

Replication initiation in Bacteria: step 1

In E. coli, three enzymes, DnaA, DnaB, and DnaC, bind oriC and initiate replication

Replication initiation in Bacteria: step 2

DnaA binding to part of the oriC sequence results in unwinding of DNA

Replication initiation in Bacteria: step 3

to stabilize the single strands of DNA, SSB (single stranded binding protein) binds to the unwound regions

Replication initiation in Bacteria: step 4

DnaB is a DNA helicase, which unwinds the DNA strands as replication proceeds

DNA polymerase

an enzyme that can copy DNA molecules

Incoming nucelotides are added to the 3’ hydroxyl end of the DNA…

chain, so elongation occurs in the 5’ to 3’ direction

original DNA polymerase form is

DNA polymerase I

Okazaki fragments

the discontinuous fragments that the lagging strand is synthesized by

the leading strand is synthesized as…

a continuous chain

Reiji Okazaki isolated DNA from bacteria that were…

briefly exposed to a radioactive substrate incorporated into newly made DNA

Much of the radioactivity in Okazaki’s experiment were..

located in small fragments about 1000 nucleotides long

With longer labeling, the radioactivity became associated with….

longer molecules, this conversion did not take place in bacteria lacking DNA ligase

About 1 of every ______ nucleotides incorporated during DNA replication is ____

100,000 ; incorrect

All of the incorrect nucleotides are usually fixed by..,

a proofreading mechanism

Almost all DNA polymerases have a …

3’ → 5’ exonuclease activity

Exonucleases

degrade nucleic acids from the ends of the molecules

Endonucleases

make internal cuts in nucleic acid molecules

The exonuclease activity of DNA polymerase allows it to….

remove incorrectly base-paired nucleotides and incorporate the correct base

DNA polymerase can add nucleotides only…

to the 3’ end of an existing nucleotide chain

Researchers implicated RNA in the initiation process based on observations: observation 1

Okazaki fragments usually have short stretches of RNA at their 5’ ends

Researchers implicated RNA in the initiation process based on observations: observation 2

DNA polymerase can add nucleotides to RNA chains as well as DNA chains

Researchers implicated RNA in the initiation process based on observations: observation 3

cells contain an enzyme called primase that synthesizes short (10 bases) chains of RNA using DNA as a template

Researchers implicated RNA in the initiation process based on observations: observation 4

primase is able. to initiate RNA strands without a preexisting chain to add to

DNA synthesis is initiated by the formation…

of short RNA primers

the short RNA primers are synthesized by…

primase using a single DNA strand as the template

During DNA replication, the two strands of the….

double helix must unwind at each replication fork

Three classes of proteins facilitate the unwinding:

• DNA helicases

• topoisomerases

• single-stranded DNA binding proteins

DNA helicase

responsible for unwinding the DNA, using energy from ATP hydrolysis

Topoisomerase

relieve the supercoiling caused by helix unwinding by making and quickly sealing double-stranded or single-stranded breaks

SSBs (single-stranded DNA binding proteins)

keep the DNA unwound and accessible to the replication machinery

starting at the origin of replication…

the machinery at the replication fork adds proteins required for synthesizing DNA

the proteins involved in replication

• DNA helicase

• DNA gyrase

• SSB

• primase

• DNA polymerase

• DNA ligase

The proteins involved in replication are closely…

associated in a large complex called a replisome

Replisome

• the size of a ribosome

• complex of proteins that are involved in replication

Activity and movement of replisome

is powered by nucleoside triphosphate hydrolysis

As the replisome moves along the DNA, it must…

accommodate the fact that DNA is being produced on both leading and lagging strands

trombone model

the model for how the replisome works

• a sliding clamp protein that attaches to a DNA polymerase catalytic subunit allows the polymerase to “process” along the DNA without falling off

In eukaryotes, replication of linear chromosomes is initiated…

at multiple sites, creating replication units called replicons

DNA of a typical chromosome may contain several ….

thousand replicons, each 50,000 to 300,000 bp in length

At each origin of replication, two replication forks…

synthesize DNA in opposite directions, forming a “replication bubble”

Replication initiation in eukaryotes: step 1

origins of replication recruit proteins that initiate the unwinding and replication of DNA

Replication initiation in eukaryotes: step 2

a multisubunit protein complex called the origin recognition complex (ORC) binds the replication origin

Replication initiation in eukaryotes: step 3

the minichromosome maintenance (MCM) proteins bind the origin

• include DNA helicases and helicase loaders

Replication initiation in eukaryotes: step 5

all the DNA-bound proteins make up the pre-replication complex, and the DNA is “licensed” for replication

replication factories

immobile structures that synthesize DNA as chromatin fibers are fed through them

chromatin remodeling proteins

facilitate the unfolding of chromatin fibers ahead of the replication fork, this loosens nucleosome packing

After a stretch of DNA is replicated, nucleosomes…

are reassembled on the newly formed strands

The dynamic disassembly/reassembly allows…

nucleosome association with DNA throughout the replication process

Linear DNA molecules have a problem in completing…

DNA replication on the lagging strand because primers are required

Each round of replication would end with the…

loss of some nucleotides from the ends of each linear molecule

telomeres

highly repeated sequences at the ends of chromosomes which solved the DNA End-replication problem

these noncoding sequences (telomeres) ensure that the cell….

will not lose important genetic information if DNA molecules shorten during replication

telomerase

a polymerase that can catalyze the addition of repeats to chromosome ends

What is telomerase composed of?

protein and RNA

In the protozoan Tetrahymena, the RNA component of the…

telomerase (3’—AACCCC—5’) is complementary to the telomere repeat sequence (5’—TTGGGG—3’)

the enzyme-bound RNA (telomerase) acts as a template for….

adding the DNA repeat sequence to the telomere ends

After telomeres are lengthened by telomerase…

telomere capping proteins bind to the exposed 3’ end to protect the degradation

In many eukaryotes, the 3’ ends of the DNA also…

loop back and base-pair with the opposite strand to form a protective closed loop

In multicellular organisms, telomerase function is…

restricted to germ cells and a few other types of actively proliferating cells

telomere shortening occurs with each cell division in most cells, as a result…

telomere length is a counting device for how many times a cell has divided; if a cell divides too many times, telomeres could be lost

cells at risk of loss of telomeres undergo…

apoptosis, programmed cell death

Immortalized cell lines, such as HeLa cells…

produce telomerase and ca be passaged indefinitely

Cell death triggered by a lifetime of telomere shortening is though to…

contribute to some of the degenerative diseases associated with human aging

Scientists speculate that telomerase-based therapy may one day…

be used to combat symptoms of human aging

Proteins that bind the tandem repeat DNA in telomeres recruit…

telomere capping proteins to protect the single-stranded DNA at the ends from damage

Patients with Werner syndrome lack….

a telomere cap protein (WRN) and exhibit premature signs of aging

DNA alterations, or mutations, can arise spontaneously or ….

through exposure to environmental agents

During DNA replication, some types of mutations occur through…

1. spontaneous mispairing of bases due to transient formation of tautomers

2. slippage during replication

3. spontaneous damage to individual bases

Mispairing of DNA nucleotides due to presence of…

tautomers is the most common form of spontaneous replication error

Tautomers are rare, alternate…

resonance structures of nitrogenous bases

tautomeric shift

a base can pair in a nonstandard way, the result is a new daughter strand that carries an incorrect base at that position

trinucleotide repeats

• example of spontaneous replication error that can occur in regions with repetitive DNA

• they are susceptible to strand slippage

• DNA polymerase replicates a short stretch of DNA twice

depurination

the loss of a purine base

deamination

the removal of a base’s amino group

failure to repair depurination and deamination can lead to…

base changes in the DNA sequence

mutagens

mutation-causing agents

Environmental mutagens

• chemicals

• radiation

Mutation can also be induced by mobile genetic elements, such as…

found in viruses, or transposable elements (transposons)

Base analogues

resemble nitrogenous bases and are incorporated into DNA

• wherever it is incorporated into DNA, when the DNA is replicated, an A is incorporated into the new strand

Base-modifying agents

react chemically with DNA bases to alter their structures, forming DNA adducts

Intercalating agents

insert themselves between adjacent bases, distorting DNA structure

• when repaired, there may be additions or deletions of nucleotides

Ultraviolet radiation alters DNA by triggering…

pyrimidine dimer formation - covalent bonds between adjacent pyrimidine bases

X-rays and related types of radiation, called ionizing radiation do what?

remove electrons from molecules and generate highly reactive intermediates that damage DNA