DNA replication

Replication or reduplication refers to the duplication of the genetic information carrier DNA in a cell of a semi- conservative (from the Latin semi " half"; conservare " get " ) principle. This is usually an exact duplication of the DNA. The replication is initiated usually only in a specific phase of the cell cycle: In eukaryotes during the synthesis phase, and S phase, prior to mitosis, ie before a mitosis. Since the genetic doubling together with the cellular doubling represents the multiplication of prokaryotes, so all bacteria and archaea, the replication is strongly coupled with cell division. Viruses use the necessary raw materials and enzymes of the host cell.

The reproduction is done semi- conservative, that is, the original DNA double strand is separated into its single strands, where then each complementary strands are newly formed. " Complementary " means that a single strand of DNA determines the base sequence ( sequence ) of the opposite strand clearly, because each base of a DNA nucleotide can only with a fixed partner via hydrogen bonds a stable relationship go (adenine - thymine and guanine - cytosine, see also base pairs ).

Graph

Phases

The sequence of replication can be divided into three phases:

Mechanisms

The replication can proceed by several mechanisms, of which the following are known:

• The reproduction of the nuclear genome of eukaryotic cells as well as of the bacterial genome is " bidirectional" instead, so the replication from the starting point, starting is done ( the start sequence is usually in the middle of the string, not the beginning ) in both directions, on both mother strands simultaneously. The bi-directional principle is the principle most frequently found in nature.
• The reproduction according to the " rolling circle " principle. It occurs primarily in the conjugation, in which a bacterium is a further single-strand of a plasmid to another.
• The " asymmetric replication " the DNA of the mitochondria ( mtDNA), also called "D -loop " in which replication takes place at the parent strands are not simultaneously but time-shifted.

Semi conservative principle

The replication after the semi- conservative principle was already postulated by Watson and Crick, as it is derived from the DNA double -stranded structure of genetic information. In addition to two other principles ( conservative and dispersive principle) the semi-conservative was occupied by the Meselson -Stahl experiment. The basic principle states that the original double strand is opened and used, both partial strands as a template for replication. In this case, the replicated strand is again formed according to the Watson -Crick base pairing. The replication after the semi- conservative principle represents the general scholarly replication mechanism corner shows other principles are special cases that are only partially proved.

Since all bacteria as well as the nucleus of all eukaryotic cells contain double-stranded DNA replication mechanism of this is found in nature and most commonly used. Exceptions are some mitochondria, which runs a different mechanism, as well as plasmid and viral genomes, in which the genetic information can also be present as single-stranded DNA. Here, a completely different mechanism must be found. In retroviruses whose genetic information is always present in the form of an RNA double or single strand, replication is performed by the host cell by the RNA is transcribed by reverse transcriptase into DNA and incorporated into the host genome.

Replication in prokaryotes

Initiation

In the cell, the helical DNA is not arranged circular or linear ago, but is also twisted in itself (so-called " supercoils " DNA double helix). In order to be able to replicate, it must be unwound. The result of the unwinding of the DNA at a point is the increasing twisting of the entire DNA double strand. To counteract torsional stresses in the unwinding run before each replication fork, a topoisomerase, which can reduce the twist ( relaxation response ). For this, the cleavage of the DNA strands is necessary. Depending on the type of enzyme controlled single or double strand breaks can be performed. After the unwinding of the previously split phosphoric acid ester bonds of the sugar phosphate backbone of the DNA by the enzyme may be linked again.

For the initiation of replication, a special place, the origin of replication on the circular DNA is usually required, which determines the start point. At this point, the hydrogen bonds between the bases of the two single strands are separated.

The so-called oriC comprises 245 base pairs ( bp ) and contains a tandem arrangement with AT -rich sequences, the consensus sequence:

5'- GATCTNTTNTTTT -3 '                    3'- CTAGANAANAAAA -5 ' and 5 binding sites for the DnaA protein. First, the initiator protein DnaA is activated by ATP and bound to five 9 -bp DnaA boxes. Overall, about 20 DnaA proteins are clustered together as a loop around the DNA. The proteins IHF and FIS bind to specific sections of the oriC and solve the diffraction of DNA into a hairpin- like structure, which also supports the binding of DnaA. Finally, the unwinding of the double helix to three consecutive (also called " 13mer sequences" ) 13 bp adenine - and thymine -rich sequences start.

This process is catalyzed by a helicase with ATP consumption. E. coli DnaB and this is passed through the protein dnaC to Origin. Due to the separation of the double strand arise as to the Origin two replication forks that diverge bidirectionally during replication. Thus, the base pairs not again via hydrogen bonds, keep so-called single-strand binding proteins ( in prokaryotes, this means " SSB protein " for engl. "Single- beach -binding protein" ), the individual strands apart.

Following the opening of the priming follows: To the now free single strands is set by an RNA polymerase, primase, a short piece of RNA, the primer ( about 10 nucleotides, under cellular conditions ). This complex is referred to as Primosom and is necessary because of the major protein complex of replication, the DNA polymerase with the synthesis of the second strand DNA in each case can only start at a free 3' -OH group. This means that the DNA polymerase requires the primer as a " jump start " for replication, although this is RNA. The primer used for the RNA polymerase requires only the single strand as a template., The DNA polymerase but then started with the synthesis of the second strand ( from 5 'to 3'), it can hardly be broken, and working up to the termination continuously. Thus, the regulation of replication must happen in the initiation phase.

Elongation

After initiation, and it enters its polymerization, the elongation phase is run. Here, the DNA polymerase synthesizes the complementary strands to the single strands: The bases of the individual strands are successively read and, on the principle of base-pairing, mounted according to the synthesized strand sequentially. For DNA synthesis Blocks which are in the form of free nucleotides in the cell.

However, this causes a problem: the DNA polymerase can only synthesize in the 5 ' to 3' direction. However, since the DNA polymerase complex synthesized simultaneously on both strands, both strands are oriented but opposite according to the double-helical structure results in two opposite directions ( antiparallel ) strands at the replication fork. A distinction is made between the leading strand (including the forward strand or continuous strand, engl. "Leading beach " ), which is seen from the replication fork oriented in the 5' -3 'direction, and the lagging strand (also reverse strand or discontinuous strand, engl. "lagging beach " ) ( 5'- 3 'direction also, but on the other strand ). It should be noted that the newly synthesized DNA strands present in the opposite orientation as their respective template strands.

On the leading strand can after a single priming to the symmetry axis of the replication fork are replicated continuously, because it is oriented exactly with the reading direction of the polymerase complex and in the direction of the replication fork. On the lagging strand continuous replication, however, is not possible because he was " wrong direction " runs in the. The polymerase runs in the first run to the primer of the second Leitstranges replication fork, which extends in the other direction. After the interruption, re- priming on the lagging strand must be made so that the polymerase can begin again again. This priming is always direct the helicase below. In the following cycles, on the lagging strand polymerase irregular polymerase terminates the synthesizing on the last RNA primer, that is, at the 5 ' end of the previous fragment. The resulting DNA fragments are referred to as individual Okazaki fragments.

Since the polymerase complex in only one direction, namely behind the helicase forth runs, the lagging strand must be aligned accordingly. There are some well-documented evidence that the area between primase and the last Okazaki fragment is twisted as a loop, so that the polymerase can edit both strands with the same direction. Is the synthesis of the loop ends, this is again dissolved and formed a new loop. Here again appears to contribute a topoisomerase.

Since the doubling of a continuous strand, the other runs intermittently, one also speaks of a semidiskontinuierlichen doubling.

In order that a continuous strand is produced, which does not contain RNA fragments, or another mechanism comes into action during replication: an RNase H from the RNA primer and a further DNA polymerase ( in prokaryotes and eukaryotes, this is, the DNA polymerase I) fills the resulting gap with the respective complementary DNA.

The DNA ligase then closes the binding of the 3 ' end of the new to the 5'- end of the old piece of DNA, thus provides between the newly synthesized DNA strands, the phosphodiester bonds forth.

Termination

In the prokaryotes with an annular constructed DNA termination have been found with respect to the Origin location. It is more accurate to two sequences, one for each replication fork. Normally, the termination must be not particularly triggered because when two replication forks on each other or run the DNA, as in a linear form, ends, replication is stopped automatically. This is a control element, so that the speeds of the two replication replicate at different replication forks controlled at a certain point ends. The termination sites are binding sites for the protein Tus ( terminus Utilizing substance ). This blocks the replicative helicase ( DnaB ), thus bringing to a halt the replication. The replicated annular strands remain in prokaryotes after replication still for a while interconnected, exactly at this terminal site so that they can be finally separated and divided according to the division of other processes. Without that connection, a control in the distribution does not seem to be present. The separation of DNA circles can take place via two mechanisms whereby either a type I or type II, a topoisomerase is involved.

Replication in eukaryotes

The replication runs in eukaryotes mainly from identical. However, there are some exceptions and special cases. It must be considered that the DNA is more "packed" ( for example, when heterochromatin ), the DNA -binding proteins have ( histone and non-histone proteins) greater influence and the DNA is present in a linear form. Moreover, it is among the proteins involved in typically those with the same functionality, but different structure.

One of the main differences lies in the Initiation: In eukaryotes there are several Origins. The advantage of this is readily apparent, firstly because the replication is slower by more existing DNA-binding proteins, on the other eukaryotic polymerase, which has (so-called " proofreading " ), slowly progresses a more complex repair mechanism than in prokaryotes. The polymerase creates in eukaryotes about 50 to 100 nucleotides per second, whereas in prokaryotes more than 1000 nucleotides can be added per second. Further, the eukaryotic DNA is significantly greater than that of prokaryotes usually ( a few million in prokaryotes over a few million base pairs in eukaryotes ). More origins and thus more replication units reduce the time required to replicate the entire genome, even if the speed of prokaryotes is not achieved ( the replication of prokaryotes is in the range of a few minutes, in eukaryotes, it is several hours).

The Origins of eukaryotes have no particular sequence, but it is probably doing a so-called consensus sequence, ie a sequence similarity. These are also known as ARS elements. Other findings assume that large regions of DNA called replication centers, can serve as a possible starting points for replication. Which, as always, as detected Origin posts are made by an Origin recognition complex ( ORC, origin recognition complex ), a Cdc6 protein and so-called MCM proteins ( minichromosome maintenance protein), which serve as helicases marked. This quasi later removed proteins form the vanguard for replication. The ORC binds to the origin, continue to recruit ORC other factors ( Cdc6, Cdt1 and helicase loading protein ), then bind MCM helicases, which melt the DNA. Only once during the S- phase replication is initiated (despite the 10,000 Origins on the eukaryotic genome).

The further course of initiation and elongation is with the prokaryotes functionally identical.

Terminal sequences have not been detected in eukaryotes. You also seem to have no meaning, because the replication machinery is terminated automatically when the end of the DNA is achieved. These results, however, in contrast to the circular DNA structure of prokaryotes, a problem: the DNA polymerase synthesizes the parental strand each of 3 'to 5 '. ( So the daughter strand, the orientation 5' -3 ') However, the polymerase requires a primase jumpstart to duplicate DNA can. Primase is an enzyme which replicates a short starting sequence of the DNA and RNA. This initial part of the present DNA polymerase, a nucleotide with a free 3'-OH end at which it can continue to synthesize DNA nucleotides. After the successful synthesis of the RNA primers are destroyed by enzymes, thus leaving gaps. At the telomeres, the ends of chromosomes, but these gaps can not be closed because no previous 3'- end is available. Similarly, the Okazaki fragments are formed during synthesis of the delay train. Here, the polymerase must work namely in the wrong direction, which is why many primases must be used. However, the resulting gap can be combined with DNA ligases. This is possible because there is always a preceding nucleotide present at a 3'-end. Since no such nucleotides are present at the telomeres is not possible to complete the synthesis of the ends. So be reduced for each chromosome doubling the telomeres at the 5'- end of both strands daughters. Since telomeres are composed of a tandem - repetitive sequence that is behind the other of a repeating sequence which does not involve structural genes, is a loss to a certain length is not a great disadvantage. But one suspects that the DNA replication with increasing number of unstable, since the stabilizing effect of the telomeres is becoming weaker. Maybe this could be a genetic indicator of aging.

In germ cells and stem cells as well as in some tumor cells have been discovered an enzyme called telomerase, which compensates for this loss. This is a reverse transcriptase, an RNA-dependent DNA polymerase, it is in the repetitive sequence as a template before. It extends the leading strand to some sequences so that the DNA polymerase can synthesize the sequence after completion of the priming strand.

During S phase of the cell cycle, the protein binds the two sister chromatids cohesin the entire length to one another. During anaphase the cohesin the enzyme separase dissolves again, so that the sister chromatids are pulled out of the spindle fibers to the cell poles.

Rolling circle principle

In viruses and plasmids occurs at a different replication principle: If it is circular double-stranded DNA, one strand is broken by an endonuclease, that is, here the connection between two adjacent bases is interrupted on one strand. At this break point is then also set to a DNA - polymerase complex, but operates only in one direction. The 3'-OH end of the cut strand is used as a starting point (so-called "primer "). At the breaking point of the broken strand is polymerized further, this strand is thus extended. Here, the unbroken complementary strand serves as a template. Since the unit of replication by wandering around the inner strand as a rolling circle, one speaks of the Rolling -circle principle.

The inner strand may repeatedly serve as a template so that multiple duplicates can be consecutively synthesized. These are broken down after the first replication step after another and used as template for the second step, setting out the replicated double-stranded DNA.

It is the single-stranded DNA as before, before the DNA is transformed into a double-stranded DNA.

The Rolling -circle principle seems to exist in nature in various forms. The exact course has not been elucidated so far. The procedure described represents the most common hypothesis

Asymmetrical principle

The replication of mitochondrial DNA and plastider has a difference in the end: The polymerase replicates delayed, it begins replication on one strand and only when the already replicated strand has displaced more than two-thirds of the original DNA, it dissolves completely and the dissolved strand is independently replicated. The polymerase also replicates it in only one direction.

This principle is also called the D -loop or displacement loop.