(L 2, 3) DNA Replication in Prokaryotes and Eukaryotes. (Dr. Carmen Cadilla)

In eukaryotic cells, DNA is found associated with various types of proteins (known collectively as nucleoprotein) present in the nucleus. o Each chromosome in the nucleus of a eukaryote contains one long, linear molecule of dsDNA,

which is bound to a complex mixture of proteins (histone and non-histone) to form chromatin Chromatin = complex of DNA + protein

o Eukaryotes have closed, circular DNA molecules in their mitochondria. o In prokaryotes, the proteinDNA complex is present in a nonmembrane-bound region known as

the nucleoid. Prokaryotic cells: lack nuclei, have a single chromosome, and may also contain

nonchromosomal DNA in the form of plasmids. Plasmids may carry genes that convey antibiotic resistance to the host bacterium, and

may facilitate the transfer of genetic information from one bacterium to another. The plasmid contains an origin of replication and associated DNA regulatory sequences

that are together called a replicon A prokaryotic organism typically contains a single, double-stranded, supercoiled, circular

chromosome. DNA exists as a double-stranded (ds) molecule, in which the two strands wind around each other,

forming a double helix. *Con la excepcion de unos cuantos viruses q tienen ss-DNA, el DNA es por lo general ds-DNA. o DNA double helix:

Each DNA molecule is composed of two polynucleotide chains joined by hydrogen bonds between the bases

The chains are antiparallel: one chain runs in a 5 to 3 direction and the other chain runs 3 to 5

Base pairing: The base pairs are held together by hydrogen bonds: 2 between A and T and 3 between

G and C These hydrogen bonds, plus the hydrophobic interactions between the stacked bases,

stabilize the structure of the double helix. Purines & Pyrimidines:

o Pyrimidines are C U T o Purines G, A

o Phosphodiester linkages between nucleotides (in DNA or RNA) can be cleaved hydrolytically by chemicals, or hydrolyzed enzymatically by a family of nucleases: deoxyribonucleases for DNA and ribonucleases for RNA

o Nucleosides can be phosphorylated by specific kinases in the cell on the sugar's primary alcohol group (-CH2-OH) to produce nucleotides. Nucleotides are the molecular building-blocks of DNA and RNA.

Semiconservative Model = The double-stranded DNA contains one parental and one daughter strand following replication

DNA is thought to consist primarily of B-DNA, which is right-handed and contains10 base pairs per turn. o Other forms of DNA include the A form, which is similar to the B form but more compact, and

the Z form, which is left-handed and has its bases positioned more toward the periphery of the helix.

Central Dogma of Molecular Biology: o DNA RNA proteins

Replication, transcription, translation Replication in prokaryotes = DNA replication begins at a single, unique nucleotide sequencea site

called the origin of replication. Replication in eukaryotes = begins at multiple sites along the DNA helix DNA replication relies on the complementarity of DNA strands = AT/GC rule (Chargaffs rule) The enzymes involved in the DNA replication process are template-directed polymerases that can

synthesize the complementary sequence of each strand with extraordinary fidelity. Prokaryotic Replication: As the two DNA strands unwind and separate, they form a V where active synthesis occurs, a

region called the replication fork. As the two strands of the double helix are separated, positive supercoils are produced in the region

of DNA ahead of the replication fork. DNA Supercoiling:

o Positive supercoiling of DNA occurs when the right-handed, double-helical conformation of DNA is twisted even tighter (twisted in a right-handed fashion) until the helix begins to distort and "knot."

o Negative supercoiling, on the other hand, involves twisting against the helical conformation (twisting in a left-handed fashion), which preferentially underwinds and "straightens" the helix at low twisting stress, and knots the DNA into negative supercoils at high twisting stress.

Linking number in relaxed DNA: # of base pairs divided by the # of base pairs per helical turn. o Example: Relaxed circular dsDNA of 2,100 base pairs in the B form (10.5 base pairs per turn) has

linking number of 200. Replication of dsDNA is bidirectionalthat is, the replication forks move in opposite directions from

the origin, generating a replication bubble. 3 types of DNA sequences in oriC are functionally significant:

o AT-rich region o DnaA boxes o GATC methylation sites

Initiation of DNA replication requires the recognition of the origin of replication by a group of proteins that form the prepriming complex: **Example used is E. coli, with oriC (origin of Chromosomal replication) o DnaA protein: binds to specific nucleotide sequences at the origin of replication, causing short,

tandemly arranged (one after the other) AT-rich regions in the origin to melt. DnaA is a protein that activates initiation of DNA replication in bacteria. It is a replication

initiation factor, which promotes the unwinding of DNA at oriC. The binding of DnaA protein to its DNA binding sitesDnaA boxesin the chromosomal

oriC region is essential for initiation of chromosome replication. In E. coli, oriC is bound by an initiator protein called DnaA. DnaC then associates and acts

like a matchmaker to allow DnaB, the helicase, to bind and begin separating the parental strands to create a replication fork. A replication bubble is formed at each origin and a pair of replication forks is established that move away from the origin, one in each direction. Thus, replication is bidirectional.

o GATC methylation sites: Before replication, GATC sites are methylated on both strands. This full methylation facilitates initiation of DNA replication.

Following replication, GATC sites are not methylated on the daughter strands (hemimethylated).

This half-methylation does not efficiently initiate replication. Several minutes will pass before Dam methylase will methylate the GATC sites in the daughter strands

DNA helicases: bind to ssDNA near the replication fork, and then move into the neighboring double-stranded region, forcing the strands apartin effect, unwinding the double helix by breaking the hydrogen bonds between the 2 strands, creating 2 replication forks (bidirectional replication). o Composed of 6 subunits o Travels along the DNA in the 5 to 3 direction o Uses energy from ATP

Single-stranded DNA-binding (SSB) proteins: bind to the ssDNA generated by helicases. o keep the 2 strands of DNA separated in the area of the replication origin, thus providing the

single-stranded template required by polymerases, and also protect the DNA from nucleases that degrade ssDNA.

DNA topoisomerases: responsible for removing supercoils in the helix. o 2 major types of topoisomerases:

Type I topoisomerases: work by making a transient cut in one DNA strand Type IA topoisomerases:

o break one strand o connect broken ends to the enzyme o pass the intact one through the gap o join the ends of the broken strand o change linking number strictly in increments of one

Type IB topoisomerases: o break one strand o allows the free cleaved strand to rotate around the other o join the strands o change linking number by more than one

Type II topoisomerases: work by making a transient cut in both DNA strands o break both strands o pass a DNA segment through the break o join the strands o change linking number in increments of 2

o DNA gyrase, often referred to simply as gyrase, is an enzyme that relieves strain while double-strand DNA is being unwound by helicase. It is also known as DNA topoisomerase II

o Topoisomerase I = mammalian nicking-closing enzyme Direction of DNA Replication:

o The 2 new daughter strands are synthesized in different ways: Leading strand:

One RNA primer is made at the origin DNA pol III attaches nucleotides in a 5 to 3 direction as it slides toward the opening of

the replication fork Lagging strand:

Synthesis is also in the 5 to 3 direction However it occurs away from the replication fork Many RNA primers are required

DNA pol III uses the RNA primers to synthesize small DNA fragments (1000 to 2000 nucleotides each)

These are termed Okazaki fragments RNA primer:

o DNA polymerases cannot initiate synthesis of a complementary strand of DNA on a totally single-stranded template. Rather, they require an RNA primerthat is, a short, double-stranded region consisting of RNA base-paired to the DNA template, with a free hydroxyl group (-OH) on the 3'-end of the RNA strand.

o DNA primase (DnaG) synthesizes the short stretches of RNA (~10 nucleotides long) which serves as primer

o Primosome: makes the RNA primer required for leading strand synthesis, and initiates Okazaki fragment formation in lagging strand synthesis.

o DNA helicase + DNA primase = primosome complex o primosome is physically associated with the DNA polymerase holoenzyme forming the

replisome Chain elongation:

o Prokaryotic (and eukaryotic) DNA polymerases elongate a new DNA strand by adding deoxyribonucleotides, one at a time, to the 3'- end of the growing chain

o DNA polymerases catalyzes a phosphodiester bond between the innermost phosphate group of the incoming deoxynucleoside triphosphate & 3-OH of the sugar of the previous deoxynucleotide

o In the process, the last 2 phosphates of the incoming nucleotide are released in the form of pyrophosphate (PPi)

DNA pol I & DNA pol III = normal replication o DNA pol I: removes the RNA primers and fills the resulting gap with DNA

1. Uses 5 3 exonuclease activity to digest the RNA 2. Uses 5 3 polymerase activity to synthetize DNA 3. Uses 3 5 exonuclease activity for proofreading

o DNA pol III: remains attached to the template as it is synthesizing the daughter strand (processive feature) Processive means it remains bound to the template strand as it moves along, and does not

diffuse away and then rebind before adding each new nucleotide. This processive feature is due to several different subunits in the DNA pol III holoenzyme

subunit is in the shape of a ring. It is termed the clamp protein subunit is needed for to initially clamp onto the DNA. It is termed the clamp-loader

protein DNA pol III is composed of 10 subunits, which all together are known as DNA pol III

holoenzyme The alpha subunit synthesizes DNA

DNA pol III continues to synthesize DNA on the lagging strand until it is blocked by proximity to an RNA primer. When this occurs, the RNA is excised and the gap filled by DNA pol I.

In the absence of the subunit DNA pol III falls off the DNA template after a few dozen nucleotides have been polymerized

In the presence of the subunit DNA pol III stays on the DNA template long enough to polymerize up to 50,000 nucleotides.

DNA pol II, DNA pol IV, DNA pol V = DNA repair & replication of damaged DNA

Termination of Replication: Opposite to oriC is a pair of termination sequences called ter sequences, designated T1 and T2 o The protein tus (termination utilization substance) binds to these sequences and it can then

stop the movement of the replication forks o DNA replication ends when oppositely advancing forks meet (usually at T1 or T2) o Finally DNA ligase covalently links all 4 DNA strands o DNA replication often results in 2 intertwined molecules

Intertwined circular molecules are termed catenanes These are separated by the action of topoisomerases

o Proofreading mechanisms: Mistakes during the process are extremely rare 3 main reasons why fidelity is high:

o Instability of mismatched pairs o Configuration of the DNA polymerase active site o Proofreading function of DNA polymerase 3 5 exonuclease activity.

Eukaryotic DNA Replication

Multiple origins of replication o Tandemly arrayed along the chromosomes (50k-100k bp intervals) o Proceeds bidirectionally o Origin Recognition Complex (ORC)

A six subunit complex that acts as the initiator of replication (ATP required) ORC binds to the origin. MCM complex also binds to this, with the help of cdc6 protein. The initiator complex is activated and the helicase activity opens the parental

strands, forming a small bubble. SSB binds to exposed single strands, helicases are loaded onto the DNA, and the

bubble is enlarged. Pol /primase synthetizes the first RNA primer containing 10 nt., the switches to elongating

with deoxyribonucleotides, and adds about 15-30 nt. (RNA-DNA hybrid is formed). Pol/primase leaves and

The chain is then elongated by polymerase , which incorporates deoxyribonucleotides to the strand.

Eukaryotic DNA Polymerases

DNA pol/primase creates the RNA-DNA hybrid that is used by DNA pol or for the

processive elongation of the leading and lagging strand (processive polymerases). This exchange is known as polymerase switch. Eukaryotic Lagging Strand Synthesis

o Helicase activity separates parental strands o Single-strand DNA binding protein called RPA binds to exposed strands o Pol/primase complex initiates the Okazaki fragment by synthetizing the RNA-DNA

hybrid primer. o When the hybrid is synthesized, pol/primase complex dissociates and RPC binds to this

elongated primer and serves as a clamp loader to assemble the PCNA sliding clamp. o Pol binds the PCNA and completes Okazaki fragment to final length of 130-200bp

Further Okazaki Fragment Processing o Primer removal is carried is carried out in two steps by RNase H and FEN1

RNase H degrades the RNA primer, leaving a single ribonucleotide attached to the end of the Okazaki fragment.

FEN1 removes the last ribonucleotide (and some deoxyribonucleotides) and forms a small flap of a few nucleotides which are cleaved at the angle of the flap

o If pol mismatched the first few nucleotides synthetizing the Okazaki fragment, a larger flap is created by destabilization and FEN1 excises it. This increases the accuracy of repl.

o The remaining gap is filled by pol . DNA pol plays a role in Base Excision Repair (BER) Lesion Replicating Polymerases are involved in replication of damaged DNA. Nucleosomes and DNA Replication

o More histones are synthesized when DNA is being actively replicated. (S phase) o They associate with the newly made DNA near the replication fork o Daughter strands contain mixture of old and new histones

Telomeres and DNA replication o Telomere = telomeric DNA sequences + bound proteins o Telomere replication problem there is no place to synthesize a primer that would

allow the daughter strand to be completed, so the daughter strand will be shorter than the parental strand.

o Telomeric sequences moderately repetitive tandem arrays of 6 nt sequence (TTAGGG) 3 overhang 12-16 nt. long

o Telomerase a ribonucleoprotein (RNA-containing) Short RNA strand that partly base-pairs with the telomeric sequences and serves

as the template for the reaction. Protein component serves as a reverse transcriptase, synthetizing DNA from an

RNA template. It adds 6-nt repeats at a time; it can also dissociate & bind again to add 6 more

o DNA gets progressively shorter with each round of DNA replication; telomerase can elongate these ends. However, telomerase activity diminishes in the aging cell, which can cause chromosomal aberrations and eventually cell death.