DNA Replication in eukaryotic

Lecture 10 27/02/2011

11.3 EUKARYOTIC DNA REPLICATION

Eukaryotic DNA replication is not as well understood as bacterial replication
The two processes do have extensive similarities,
The bacterial enzymes described in Table 1.1 have also been found in eukaryotes

Nevertheless, DNA replication in eukaryotes is more complex

Large linear chromosomes Tight packaging within nucleosomes More complicated cell cycle regulation
Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display

11-63

QUICK REVIEW

Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display

11-37

Multiple Origins of Replication

Eukaryotes have long linear chromosomes

They therefore require multiple origins of replication

To ensure that the DNA can be replicated in a reasonable time

DNA replication proceeds bidirectionally from many origins of replication

Refer to Figure 11.20

Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display

11-64

Bidrectional DNA synthesis

Replication forks will merge

Figure 11.20

Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display

11-65

Multiple Origins of Replication

The origins of replication found in eukaryotes have some similarities to those of bacteria

Origins of replication in (Yeast)S. cerevisiae are termed ARS elements (Autonomously Replicating Sequence)

They are 100-150 bp in length They have a high percentage of A and T They have three or four copies of a specific sequence

Similar to the bacterial DnaA boxes

Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display

11-66

Multiple Origins of Replication

Origin recognition complex (ORC)

A six-subunit complex that acts as the initiator of eukaryotic DNA replication

It appears to be found in all eukaryotes

Requires ATP to bind ARS elements Single-stranded DNA stimulates ORC to hydrolyze ATP

Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display

11-67

Eukaryotes Contain Several Different DNA Polymerases

Mammalian cells contain well over a dozen different DNA polymerases

Refer to Table 11.4

Four: alpha (a), delta (d), epsilon (e) and gamma (g) have the primary function of replicating DNA

a, d and e Nuclear DNA g Mitochondrial DNA

Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display

11-68

Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display

11-69

DNA pol a is the only polymerase to associate with primase

The DNA pol a/primase complex synthesizes a short RNA-DNA hybrid

10 RNA nucleotides followed by 20 to 30 DNA nucleotides

This is used by DNA pol d or e for the processive elongation of the leading and lagging strands

Current evidence suggests a greater role for DNA pol d

The exchange of DNA pol a for d or e is called a polymerase switch

It occurs only after the RNA-DNA hybrid is made

Refer to Figure 11.21
Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display

11-70

Figure 11.21

11-71

DNA polymerases also play a role in DNA repair

DNA pol b is not involved in DNA replication It plays a role in base-excision repair

Removal of incorrect bases from damaged DNA

Recently, more DNA polymerases have been identified

Lesion-replicating polymerases

Involved in the replication of damaged DNA They can synthesize a complementary strand over the abnormal region
Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display

11-72

Nucleosomes and DNA Replication

Replication doubles the amount of DNA

Therefore the cell must synthesize more histones to accommodate this increase

Synthesis of histones occurs during the S phase

Histones assemble into octamer structures

They associate with the newly made DNA very near the replication fork

Thus following DNA replication, each daughter strand has a mixture of old and new histones

Refer to Figure 11.22

Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display

11-73

Newly made histone proteins

Figure 11.22

11-74

Telomeres and DNA Replication

Linear eukaryotic chromosomes have telomeres at both ends The term telomere refers to the complex of telomeric DNA sequences and bound proteins

Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display

11-75

Telomeric sequences consist of

Moderately repetitive tandem arrays 3 overhang that is 12-16 nucleotides long

Figure 11.23

Telomeric sequences typically consist of

Several guanine nucleotides Often many thymine nucleotides

Refer to Table 11.5

Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display

11-76

11-77

DNA polymerases possess two unusual features

1. They synthesize DNA only in the 5 to 3 direction 2. They cannot initiate DNA synthesis

These two features pose a problem at the 3 end of linear chromosomes

Figure 11.24

Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display

11-78

Therefore if this problem is not solved

The linear chromosome becomes progressively shorter with each round of DNA replication

Indeed, the cell solves this problem by adding DNA sequences to the ends of telomeres This requires a specialized mechanism catalyzed by the enzyme telomerase Telomerase contains protein and RNA

The RNA is complementary to the DNA sequence found in the telomeric repeat

This allows the telomerase to bind to the 3 overhang

The lengthening mechanism is outlined in Figure 11.25

Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display

11-79

Step 1 = Binding

The bindingpolymerizationtranslocation cycle can occurs many times This greatly lengthens one of the strands

Step 2 = Polymerization

Step 3 = Translocation

The complementary strand is made by primase, DNA polymerase and ligase

Figure 11.25

RNA primer

11-80