Replication, Maintenance, and Rearrangements of Genomic

DNA

Introduction

Reproduction is a fundamental biological process. Each time a cell divides, its entire genome must be duplicated.

Complex enzymatic machinery is required to copy the large DNA molecules. There are even mechanisms to correct replication mistakes and repair DNA damage.

But cell genomes are not static.

In order for species to evolve, mutations and gene rearrangements are needed to maintain genetic variation between individuals.

DNA Replication

DNA polymerase is the main enzyme in DNA replication. It catalyzes the joining of deoxyribonucleoside 5′- triphosphates (dNTPs).

It was first identified in 1956. It provided a biochemical basis for the mode of DNA replication that was initially proposed by Watson and Crick.

Both prokaryotic and eukaryotic cells contain several different DNA polymerases that play distinct roles in DNA replication and repair.

DNA ReplicationAll DNA polymerases share two fundamental properties:

• Synthesize DNA only in the 5′ to 3′ direction.

• Add new dNTPs only to a primer strand that is hydrogen-bonded to the template; they can’t initiate DNA synthesis from free dNTPs.

DNA ReplicationDNA replication was first analyzed by growing E. coli in the presence of

radioactive thymidine, which allowed visualization by autoradiography.

DNA molecules contained two replication forks, representing the regions of active DNA synthesis.

DNA ReplicationSince the two strands of DNA run in opposite (antiparallel) directions, only one strand

of DNA is synthesized in a continuous manner in the 5′ to 3′ direction (the leading strand).

The other (lagging strand) is formed from short (1–3 kb), discontinuous pieces of DNA that are synthesized backward.

These small pieces (Okazaki fragments) are joined by DNA ligase.

DNA ReplicationShort fragments of RNA serve as primers for synthesis of Okazaki

fragments.

RNA synthesis can initiate de novo. An enzyme called primase synthesizes short fragments of RNA that act as primers.

DNA Replication

The primers must then be removed and replaced with DNA.

In prokaryotes, RNA primers are removed by polymerase I, which acts as an exonuclease that can hydrolyze DNA in either direction.

In eukaryotic cells, RNA primers are removed by RNase H, which degrades the RNA strand of RNA-DNA hybrids, and 5′ to 3′ exonucleases.

The resulting gaps are then filled by polymerase δand the fragments joined by DNA ligase.

Figure 6.5 Removal of RNA primers and joining of Okazaki fragments

DNA Replication

Different DNA polymerases have roles at the replication fork.

Eukaryotic cells have α, δ, and ε polymerases.

Polymerase α is found in a complex with primase.

DNA ReplicationOther proteins at the replication fork:

• Proteins that bind to DNA polymerases and increase their activity and keep them bound to the template DNA.

• Sliding-clamp proteins that load polymerase onto the primer and maintain its stable association with the template, and clamp-loading proteins.

Figure 6.7 Polymerase accessory proteins (Part 2)

DNA ReplicationHelicases catalyze the unwinding of parental DNA ahead of the replication

fork.

Single-stranded DNA-binding proteins then stabilize the unwound template DNA so that it can be copied by the polymerase.

DNA ReplicationAs the DNA unwinds, the DNA ahead of the replication fork is forced to

rotate, which would cause circular DNA molecules to twist.

Topoisomerases catalyze reversible breaking and rejoining of DNA strands. The transient breaks serve as swivels that allow the two strands to rotate freely around each other.

DNA ReplicationThe enzymes involved in DNA replication act in a coordinated

manner to synthesize both leading and lagging strands of DNA simultaneously.

DNA Replication

The accuracy of DNA replication is critical to cell reproduction.

Mutation rates indicate the frequency of errors during replication is less than one incorrect base per 109 nucleotides.

This is much lower than would be predicted simply on the basis of complementary base pairing.

DNA polymerase helps select the correct bases for insertion.

Binding of correctly matched dNTPs induces conformational changes in DNA polymerase that lead to the incorporation of the nucleotide.

This increases fidelity of replication about a thousand-fold.

DNA ReplicationProofreading:

DNA polymerases require primers and catalyze the growth of DNA strands only in the 5′ to 3′ direction.

They also have exonuclease activity that can hydrolyze DNA in the 3′ to 5′ direction. When an incorrect base is incorporated, it is removed by the exonuclease activity.

DNA Replication

Origins of replication are binding sites for proteins that initiate the replication process.

In E. coli, an initiator protein binds to specific DNA sequences within the origin.

The initiator protein begins to unwind the DNA and recruits other proteins involved in DNA synthesis.

Helicase and single-stranded DNA-binding proteins then continue unwinding and exposing the template DNA, and primase initiates synthesis.

Two replication forks are formed and move in opposite directions along the circular chromosome.

Figure 6.12 Origin of replication in E. coli

DNA ReplicationMultiple origins are needed to replicate the much larger genomes of

eukaryotic cells in a reasonable amount of time.

This was demonstrated by exposure of mammalian cells to radioactive thymidine for different time intervals, followed by autoradiography to detect newly synthesized DNA.

DNA ReplicationEukaryotic origins of replication were first studied in the yeast S. cerevisiae.

They were identified as sequences that can support replication of plasmids in transformed cells.

Several such elements (autonomously replicating sequences, or ARSs) have been isolated.

DNA ReplicationFunctional ARS elements include an 11-base-pair core sequence common to

many different ARSs.

The core sequence is the binding site of a protein complex (origin recognition complex, or ORC) that is required for initiation of DNA replication.

DNA Replication

The terminal sequences of linear DNA molecules (telomeres) consist of tandem repeats of simple-sequence DNA.

They are maintained by telomerase, which catalyzes the synthesis of telomeres in the absence of a DNA template.

Telomerase is a reverse transcriptase.

It carries its own template RNA, which is complementary to the telomere repeat sequences.

Multiple copies of the telomeric repeat sequences can be generated to maintain telomeres.

DNA ReplicationThe mechanism of telomerase action was determined in 1985 in studies of the

protozoan Tetrahymena.

The RNA template allows telomerase to extend the 3′ end by one repeat unit beyond its original length.

The complementary strand can then be synthesized by the polymerase α-primase complex.

DNA Replication

Removal of the RNA primer leaves an overhanging 3′ end, which can form loops at the ends of eukaryotic chromosomes.

repeat fig 5.23 here

DNA Replication

Telomerase activity maintains telomeres at their normal length.

Most somatic cells do not have enough telomerase to maintain telomere length for an indefinite number of cell divisions.

Telomeres gradually shorten as cells age, and this eventually leads to cell death or senescence.

Several premature aging syndromes are characterized by an abnormally high rate of telomere loss; some are caused by mutations in telomerase.

Conversely, cancer cells express abnormally high levels of telomerase, allowing them to continue dividing indefinitely.

DNA Repair

Damage to DNA can occur spontaneously or from exposure to chemicals or radiation.

Damage can block replication or transcription, and lead to a high frequency of mutations.

DNA Repair

Cells have evolved two categories of DNA repair mechanisms:

• Direct reversal of the chemical reaction responsible for DNA damage.

• Removal of damaged bases followed by replacement with newly synthesized DNA (excision repair).

DNA RepairOnly a few types of damage are repaired by direct reversal.

Exposure to UV light forms pyrimidine dimers, which distorts the structure of the DNA and blocks transcription or replication past the site of damage.

DNA RepairPhotoreactivation is one mechanism of repairing UV-induced pyrimidine

dimers.

Energy from visible light is used to break the cyclobutane ring structure, reversing the dimerization reaction.

Many types of cells utilize photoreactivation but it is not universal; placental mammals lack this mechanism of DNA repair.

DNA RepairDNA damage can also result from alkylating agents—reactive

compounds that transfer methyl or ethyl groups to a DNA base.

Methylation of the O6 position of guanine forms O6-methylguanine, which forms complementary base pairs with thymine instead of cytosine.

DNA Repair

This can be repaired by an enzyme (O6-methylguanine methyltransferase) that transfers the methyl group from O6-methylguanine to a cysteine residue in its active site.

DNA Repair

Excision repair can be used for a wide variety of DNA damage.

There are three types:

• Base-excision

• Nucleotide-excision

• Mismatch repair

DNA Repair

In base-excision repair, single damaged bases are recognized and removed.

Example: uracil can arise in DNA when dUTP is incorporated in place of thymine, or uracil can be formed in DNA by the deamination of cytosine.

Excision of uracil in DNA is catalyzed by DNA glycosylase, which cleaves the bond between the uracil and the deoxyribose of the DNA.

DNA glycosylases also recognize and remove other abnormal bases.

An apyrimidinic or apurinic site (AP site) is a sugar with no base attached; formed by excision of a base.

Purine bases can also be lost spontaneously.

These sites are repaired by AP endonuclease. The deoxyribose is removed, and the resulting gap is filled by DNA polymerase and ligase.

Figure 6.21 Base-excision repair

DNA RepairOther excision repair systems recognize a wide variety of

damaged bases, including bulky groups added to DNA by carcinogens.

DNA RepairNucleotide-excision repair removes the damaged bases as part of an

oligonucleotide.

An excinuclease is an enzyme complex that can directly excise an oligonucleotide.

Helicase is required to unwind the DNA for excision; the resulting gap is filled by DNA polymerase and sealed by ligase.

DNA RepairHuman DNA repair genes have been identified in studies of

inherited diseases.

Xeroderma pigmentosum (XP) causes extreme sensitivity to UV radiation and multiple skin cancers; it results from lack of nucleotide-excision repair.

XP cells are used as an experimental system to identify DNA repair genes.

Molecular cloning has identified different repair genes that are mutated in individuals with XP and other diseases.

The encoded proteins are closely related to proteins encoded by yeast RAD genes, indicating that nucleotide-excision repair is highly conserved.

DNA Repair

The proteins are used in vitro systems to study their roles in the repair process.

Repair genes mutated in XP are designated XPA through XPG.

Disrupted base pairing is recognized by XPC, followed by the cooperative binding of XPA, RPA, and a transcription factor called TFIIH.

Two subunits of TFIIH are the XPB and XPD proteins, which act as helicases to unwind about 25 base pairs around the site of damage.

XPG protein is then recruited to the complex followed by XPF/ERCC1. These are endonucleases that cleave DNA on the 5′ and 3′ sides of the damaged region.

Figure 6.23 Nucleotide-excision repair in mammalian cells (Part 1)

DNA Repair

Transcription-coupled repair is nucleotide-excision repair during transcription.

When RNA polymerase is stalled by DNA damage in mammalian cells, it is recognized by two proteins (CSA and CSB) that then recruit other proteins to repair the damage.

In patients with Cockayne’s syndrome, genes for CSA and CSB are mutated, and they are defective in transcription-coupled repair.

Figure 6.24 Transcription-coupled repair in mammalian cells (Part 1)