Mechanisms of gene duplication and amplification

Abstract

Changes in gene copy number are among the most frequent mutational events in all genomes and were among the mutations for which a physical basis was first known. Yet mechanisms of gene duplication remain uncertain because formation rates are difficult to measure and mechanisms may vary with position in a genome. Duplications are compared here to deletions, which seem formally similar but can arise at very different rates by distinct mechanisms. Methods of assessing duplication rates and dependencies are described with several proposed formation mechanisms. Emphasis is placed on duplications formed in extensively studied experimental situations. Duplications studied in microbes are compared with those observed in metazoan cells, specifically those in genomes of cancer cells. Duplications, and especially their derived amplifications, are suggested to form by multistep processes often under positive selection for increased copy number.

Figure 1.

Duplication by exchanges between sister chromosomes. (A) a and b are sequence elements between which exchanges can be mediated by recombination, annealing, or transposition. Hybrid elements (b/a or a/b) are left at the rearrangement junctions. Duplications are subject to remodeling deletions that remove the junction. (B) Description of homologous recombination events that change copy number. These recombination events can increase or decrease copy number and make duplications prone to loss.

Figure 2.

Isolating duplications by trapping. This assay identifies and selectively maintains a preexisting duplication that formed under nonselective conditions. Frequency is defined as the fraction of recombinants that maintain two normally mutually exclusive markers. In this diagram, a Kan^R recipient receives Tet^R by a genetic cross. The duplication frequency in the recipient strain is the ratio of Tet^R Kan^R duplication transfomants to parental haploid Tet^R, Kan^S types. No amplification beyond duplication is selected. Using “recombineering” methods (Sawitzke et al. 2007), the cross that detects the duplication can be performed in recombination-deficient strains.

Figure 3.

Steady-state duplication frequencies. When a culture is started by a few cells with no duplication and is grown with no applied selection beyond viability, the duplication frequency increases to a steady state. This steady state is dictated in part by the relative rates of formation and loss (k_F and k_L). The loss rate is usually higher than the formation rate. The second factor is the fitness cost of the duplication, which is dictated by the difference between the higher growth rate of the parent strain (μ_H) and the lower growth rate of the haploid strain with the duplication (μ_D). The equation above approximates the steady-state frequency (Reams et al. 2010). In the equation, D and H denote the titer of duplication-bearing and haploid parent cells, respectively.

Figure 4.

Visual detection of chromosomal duplications and deletions. The parent strain (top line) has separated inactive lac operons (3-kb repeats) and a central active phoA gene. The parent forms a light blue colony on rich medium containing chromogenic substrates Red-Gal and Blue-Pho. An unequal exchange can generate either a duplication (center line, purple sector) or deletion (bottom line, white sector). A reciprocal exchange forms twin sectors (bottom photograph).

Figure 5.

Deletion and duplication formation in a plasmid. The tetracycline resistance determinant (Tet) is disrupted by an internal duplication formed between nascent sister chromosomes. Selection for Tet^R demands loss of the duplication by a deletion event. Drug-resistant deletion mutants carry a plasmid dimer. Although this is consistent with a reciprocal recombination, a half-exchange would produce a linear dimer, whose Tet^R phenotype could only be maintained if the plasmid recircularized by a second exchange.

Figure 6.

Formation of a tandem inversion duplication (TID). This model proposes initiation of duplication by one palindromic sequence at which a 3′ end can snap back to prime repair synthesis. Template switching to the opposite strand by this replication track would be aided by a second palindrome or closely placed inverse repeat. Resolution or replication leaves three copies of the intervening region—two copies in direct order with a central third copy in inverse order. This same process can in principle operate at a single-strand nick far from a replication fork. The product is a symmetrical TID (sTID) whose two junctions have short parental palindromes that have been extended in the sTID and may be prone to remodeling by deletion (Kugelberg et al. 2010). It is proposed that observed asymmetric join points form when deletions remove the initial palindrome and leave an asymmetric join point generated at the site of the deletion. A single large deletion that removes both junctions and the central inverse-order copy can generate a simple-tandem repeat with a short-junction (SJ) sequence. Another model achieves the same end point by template switching across two diverging replication forks (Brewer et al. 2011). The same structures can be explained by the microhomology-mediated break-induced replication (MMBIR) model described below (Hastings et al. 2009a) in which template switches are not restricted to replication fork regions.

Figure 7.

The breakage–fusion–bridge (BFB) cycle. Suggested many years ago by Barbara McClintock, this model explains the alternating orientation of copies seen in some amplification arrays. Issues are the source of the initial breaks, the forces that break a dicentric, the mechanisms of end fusions, and the stabilization of an array by blocking further end fusions. Several of these issues have been solved conceptually by the behavior of palindromic sequences.

Figure 8.

Use of palindromic sequences for induction of breaks and fusions in the breakage–fusion–bridge (BFB) model. The frequent association of palindromic sequences with amplifications in mammalian amplification suggested various ways in which they might contribute to the events in the BFB model. A break generated near a palindrome (left side, top) can leave ends whose snap-back primes repair synthesis, and serves to generate a dicentric chromosome (left side). A cruciform structure can be cut to leave snap-back ends that can similarly prime replication to form a dicentric. Heavy black lines denote duplex DNA and lighter black lines denote single strands. Ends lacking telomeres are likely to be subject to fusion and continued rounds of the cycle.

Figure 9.

Amplifications characterized in yeast and bacteria. (Top) A yeast amplification was recovered following prolonged growth selecting for increases in the SUL1 product. The rearrangement appears to have arisen between two palindromic regions. A series of five identical copies of the amplified region are present in alternating orientation (only three copies are shown). It seems likely that formation involved a snap-back structure at each palindrome and that the initial structure has not been modified by subsequent rearrangement because the junction microhomologies are in inverse order. Unequal recombination caused the further amplification from three to five copies. (Bottom) A bacterial amplification, also recovered after prolonged selection, shows different-sized repeats in opposite orientation—here, the microhomologies are not palindromic. This is proposed to have arisen following replication from snap-back structures (as seen in yeast) to form asymmetrical inversion duplication with extended palindromes at each junction. The inferred initial inversion duplication (pictured) was then stabilized by asymmetric deletions that removed the initial junction palindrome and left the observed amplification.

Figure 10.

Two views of an amplification. The diagram describes three repeats of an 11-copy amplification selected in a human cell line for amplification of the MDR1 gene (Kitada and Yamasaki 2007). (Top) The inverse-order repeats are described as identical with spacers, but can also be viewed as distinct-sized repeats because the spacers are actually continuations of the amplified region. Formation of this structure can be explained in several ways, but is consistent with deletions modifying a larger initial inversion duplication.